标签归档：脂质体

Lipo2000 Transfection Reagent 脂质体转染试剂

发表于由

描述

Lipo2000 Transfection Reagent 脂质体转染试剂

 

 

产品信息

产品名称 产品编号 规格 价格（元）         
Lipo2000 Transfection Reagent Lipo2000脂质体转染试剂             MX2211-0.15ML        0.15ml          350
Lipo2000 Transfection Reagent Lipo2000脂质体转染试剂 MX2211-0.75ML 0.75ml 950
Lipo2000 Transfection Reagent Lipo2000脂质体转染试剂 MX2211-1.50ML 1.50ml 1650
Lipo2000 Transfection Reagent Lipo2000脂质体转染试剂 MX2211-7.50ML 7.50ml 5750

 

产品描述

Lipo2000脂质体转染试剂（Lipo2000 Transfection Reagent）是一款多用途转染试剂，适用于核酸（DNA、RNA）的转染，能在绝大多数贴壁和悬浮细胞（哺乳动物细胞系）提供高效转染。独特的配方使其能直接加入培养基，血清的存在不会影响转染效率。转染后无需去除DNA- Lipo2000复合物或更换培养基，也可根据自身需求在转染4-6h后更换新鲜培养基。

 

本品以无菌液体形式提供，浓度为1mg/ml。其使用方法和Lipofectamine™ 2000 Transfection Reagent基本一致，并且经过对HEK293、HeLa等细胞的转染测试，转染效率和Lipofectamine™ 2000相当。

通常情况下，对于24孔板的DNA转染，每次用2μl左右，则1.5mlLipo2000约可转染750个孔；对于24孔板的siRNA转染，每次用1μl左右，则1.5mlLipo2000约可转染1500个孔；

保存与运输方法

保存：+4℃保存，1年有效。切勿冻存。

运输：冰袋运输。

 

注意事项

1）使用高纯的DNA或RNA有助于获得较高的转染效率，内毒素是影响转染效率高低的重要因素。

2）使用Lipo2000脂质体转染试剂需确保高细胞密度，建议转染时细胞密度达90-95%，有助于获得最高转染效率和表达水平，且能最小化高转染活性导致的细胞生长降低影响。可通过优化实验条件来降低细胞密度，但需注意不同实验间维持一个标准的接种步骤，因转染效率依赖于细胞培养密度。

 

3）转染过程中不要添加抗生素到培养基，否则会导致细胞死亡。

4）为了获得最佳的转染效率，制备转染复合物时要求用无血清培养基（比如Opti-MEM I）稀释DNA和转染试剂，因血清会影响复合物的形成。其他无血清培养基（比如DMEM）也能用来稀释DNA和转染试剂，但转染效率有可能会降低。另外，需要特别注意某些无血清配方会抑制Lipo2000介导的转染，比如CD293, SFMII, VP-SFM等。

 

5）初次使用制备复合物时，最好优化DNA（µg）和Lipo2000脂质体转染试剂（µl）的比例。DNA和Lipo2000的比例，绝大多数细胞系通常推荐1:2-1:3，比如：24孔板内接种0.5-2×105个细胞，使用0.8-1µg DNA和2-3µL Lipo2000。通过调整DNA/ Lipo2000优化转染效率很有必要。

 

6）Lipo2000脂质体转染试剂应该在4℃保存，不可冻存。使用后立即盖好盖子，避免长时间暴露在空气中，否则有可能导致脂质体氧化而影响转染效率。

7）Lipo2000脂质体转染试剂不能涡旋或离心，宜缓慢晃动混匀。

8）为了您的安全和健康，请穿实验服并戴一次性手套操作。

 

使用方法

转染步骤（以质粒DNA的24孔板为例）

【注意】：对大多数细胞来说，DNA（µg）和Lipo2000（µl）的比例为1:2~1:3。转染时高的细胞密度可以得到高的转染效率和表达水平，并能减少细胞毒性。

 

1. 细胞准备

1）贴壁细胞：转染前一天，用500 µl不含抗生素的培养基接种细胞，使之在转染时细胞达90-95%汇合（0.5~2×105 细胞/个孔，24孔板）。

2）悬浮细胞：转染当天，于准备DNA-Lipo2000复合物之前，用500µl不含抗生素的培养基接种4~8×105细胞即可。

 

2.按照以下体系配制DNA- Lipo2000脂质体转染试剂复合物：

1）对于每孔细胞，在EP管内分别加入50µl无血清培养基（比如Opti-MEM™ I Reduced Serum Medium）和0.8 µg DNA轻柔混匀，制成DNA稀释液。

2）对于每孔细胞，在EP管内分别加入50µl无血清培养基（比如Opti-MEM™ I Reduced Serum Medium）和2.0 µl Lipo2000（注意用前先混匀），轻柔混匀，制成Lipo2000稀释液，室温静置5min。【注意】：需要在30min内混合稀释的DNA和Lipo2000，更长的等待时间会降低活性。如果使用DMEM作为稀释培养基，那必须在5min内混合稀释的DNA和Lipo2000。  

3）将DNA稀释液和Lipo2000稀释液混合（总体积100µl），轻柔混匀，室温静置20min， 形成DNA-Lipo2000复合物，这一混合物可能呈浑浊状态，但不会抑制转染效率。【注意】：DNA-Lipo2000复合物在室温下至少可稳定保存5h。

 

3.将上方制备好的DNA-Lipo2000复合物加入接种好的细胞中，将培养板轻轻地前后摇动，使复合物分散均匀。【注意】：如果要在无血清条件下转染，使用含血清的正常培养基进行细胞接种。再加入复合物前吸掉培养基，更换为500µl无血清培养基。

4. 37℃，5% CO2培养箱培养24-48h，直至能进行转基因表达分析，无需去掉复合物或更换培养基。然而，有必要在4-6 h后更换生长培养基，不会降低转染活性。

 

5.特殊说明

1）对于稳转细胞株：则在转染24h后，按照1:10或更高比例接种细胞到新鲜生长培养基。转染48h后加入筛选培养基。

2）对于悬浮细胞株：在细胞中加入DNA-Lipo2000复合物后，如需要可以4h后加入PMA和/或PHA。比如：对于Jurkat细胞，分别加入PHA-L（终浓度1µg/ml）和PMA（终浓度50ng/ml），可以提高CMV启动子活性和基因表达。对于K562细胞，只加入PMA（终浓度50ng/ml）足以提高启动子活性。

 

转染体系的放大或缩小

对于不同的细胞培养板，Lipo2000、DNA、细胞和培养基的使用量根据培养表面的不同按比例进行调整，具体参考表I。对于自动化、高通量体系，以96孔板形式制备更大的复合物体积。需要注意的是，需要进行快速的96孔板转染（细胞铺板和转染同时进行），直接在平板中制备复合物，然后将细胞悬液加入到复合物内，这样进一步减少了转染时间。此种改进步骤经过293-H，293-F，COS-7L和CHO细胞的试验，同传统方法相比活性稍低。

 

表I.不同细胞培养容器中转染时培养基、核酸及Lipo2000用量

培养容器 单孔表面积*           培养基用量 DNA转染 RNAi转染
铺板培养 基用量    稀释培养 基用量**        DNA          Lipo2000   siRNA Lipo200   
96-well 0.3 cm2 100µl 2 × 25 µl 0.2 µg 0.5 µl 5 pmol 0.25 µl
24-well 2 cm2 500µl 2 × 50 µl 0.8 µg 2.0 µl 20 pmol 1.0 µl
12-well 4 cm2 1 ml 2 × 100 µl 1.6 µg 4.0 µl 40 pmol 2.0 µl
6-well 10 cm2 2 ml 2 × 250 µl 4.0 µg 10 µl 100 pmol    5 µl
60-mm 20 cm2 5 ml 2 × 0.5 ml 8.0 µg 20 µl 200 pmol 10 µl
100-mm 60 cm2 15 ml 2 × 1.5 ml 24 µg 60 µl 600 pmol 30 µl

【*】：不同厂商提供的细胞培养容器表面积可能有所不同。

【**】：稀释DNA/RNA或Lipo2000所用的无血清培养基用量。

【注意】：该表用量仅供参考，具体用量请根据细胞类型、铺板密度等其他实验条件进行优化。

 

附表II  Lipo2000脂质体转染试剂用于不同细胞转染用量参考（以96孔板为例）

细胞类型                  培养基                  每孔细胞数                 DNA用量                          Lipo2000用量                  
293H DMEM 3×104 0.2 µg 0.5 µl
293FT DMEM 3×104 0.2 µg 0.5 µl
293E DMEM 3×104 0.2 µg 0.5 µl
293F DMEM 3×104 0.2 µg 0.5 µl
HeLa DMEM 2×104 0.3 µg 0.5 µl
HepG2 DMEM 3×104 0.5 µg 0.75 µl
A549 DMEM 2×104 0.3 µg 0.5 µl
COS7 DMEM 1.5×104 0.4 µg 0.5 µl
Caco2 MEM 3.5×104 0.3 µg 0.75 µl
BHK21 MEM 2×104 0.2 µg 0.5 µl
RAW264.7 DMEM 3×104 0.2 µg 0.5 µl
CHO-K1 IMDM+Pro 3×104 0.2 µg 0.5 µl
Sf9 SIM SF 5×104 0.4 µg 0.75 µl

 

                        

Lipo2000 Transfection Reagent 脂质体转染试剂

发表于由

描述

Lipo2000 Transfection Reagent 脂质体转染试剂

 

 

产品信息

产品名称 产品编号 规格 价格（元）         
Lipo2000 Transfection Reagent Lipo2000脂质体转染试剂             MX2211-0.15ML        0.15ml          350
Lipo2000 Transfection Reagent Lipo2000脂质体转染试剂 MX2211-0.75ML 0.75ml 950
Lipo2000 Transfection Reagent Lipo2000脂质体转染试剂 MX2211-1.50ML 1.50ml 1650
Lipo2000 Transfection Reagent Lipo2000脂质体转染试剂 MX2211-7.50ML 7.50ml 5750

 

产品描述

Lipo2000脂质体转染试剂（Lipo2000 Transfection Reagent）是一款多用途转染试剂，适用于核酸（DNA、RNA）的转染，能在绝大多数贴壁和悬浮细胞（哺乳动物细胞系）提供高效转染。独特的配方使其能直接加入培养基，血清的存在不会影响转染效率。转染后无需去除DNA- Lipo2000复合物或更换培养基，也可根据自身需求在转染4-6h后更换新鲜培养基。

 

本品以无菌液体形式提供，浓度为1mg/ml。其使用方法和Lipofectamine™ 2000 Transfection Reagent基本一致，并且经过对HEK293、HeLa等细胞的转染测试，转染效率和Lipofectamine™ 2000相当。

通常情况下，对于24孔板的DNA转染，每次用2μl左右，则1.5mlLipo2000约可转染750个孔；对于24孔板的siRNA转染，每次用1μl左右，则1.5mlLipo2000约可转染1500个孔；

保存与运输方法

保存：+4℃保存，1年有效。切勿冻存。

运输：冰袋运输。

 

注意事项

1）使用高纯的DNA或RNA有助于获得较高的转染效率，内毒素是影响转染效率高低的重要因素。

2）使用Lipo2000脂质体转染试剂需确保高细胞密度，建议转染时细胞密度达90-95%，有助于获得最高转染效率和表达水平，且能最小化高转染活性导致的细胞生长降低影响。可通过优化实验条件来降低细胞密度，但需注意不同实验间维持一个标准的接种步骤，因转染效率依赖于细胞培养密度。

 

3）转染过程中不要添加抗生素到培养基，否则会导致细胞死亡。

4）为了获得最佳的转染效率，制备转染复合物时要求用无血清培养基（比如Opti-MEM I）稀释DNA和转染试剂，因血清会影响复合物的形成。其他无血清培养基（比如DMEM）也能用来稀释DNA和转染试剂，但转染效率有可能会降低。另外，需要特别注意某些无血清配方会抑制Lipo2000介导的转染，比如CD293, SFMII, VP-SFM等。

 

5）初次使用制备复合物时，最好优化DNA（µg）和Lipo2000脂质体转染试剂（µl）的比例。DNA和Lipo2000的比例，绝大多数细胞系通常推荐1:2-1:3，比如：24孔板内接种0.5-2×105个细胞，使用0.8-1µg DNA和2-3µL Lipo2000。通过调整DNA/ Lipo2000优化转染效率很有必要。

 

6）Lipo2000脂质体转染试剂应该在4℃保存，不可冻存。使用后立即盖好盖子，避免长时间暴露在空气中，否则有可能导致脂质体氧化而影响转染效率。

7）Lipo2000脂质体转染试剂不能涡旋或离心，宜缓慢晃动混匀。

8）为了您的安全和健康，请穿实验服并戴一次性手套操作。

 

使用方法

转染步骤（以质粒DNA的24孔板为例）

【注意】：对大多数细胞来说，DNA（µg）和Lipo2000（µl）的比例为1:2~1:3。转染时高的细胞密度可以得到高的转染效率和表达水平，并能减少细胞毒性。

 

1. 细胞准备

1）贴壁细胞：转染前一天，用500 µl不含抗生素的培养基接种细胞，使之在转染时细胞达90-95%汇合（0.5~2×105 细胞/个孔，24孔板）。

2）悬浮细胞：转染当天，于准备DNA-Lipo2000复合物之前，用500µl不含抗生素的培养基接种4~8×105细胞即可。

 

2.按照以下体系配制DNA- Lipo2000脂质体转染试剂复合物：

1）对于每孔细胞，在EP管内分别加入50µl无血清培养基（比如Opti-MEM™ I Reduced Serum Medium）和0.8 µg DNA轻柔混匀，制成DNA稀释液。

2）对于每孔细胞，在EP管内分别加入50µl无血清培养基（比如Opti-MEM™ I Reduced Serum Medium）和2.0 µl Lipo2000（注意用前先混匀），轻柔混匀，制成Lipo2000稀释液，室温静置5min。【注意】：需要在30min内混合稀释的DNA和Lipo2000，更长的等待时间会降低活性。如果使用DMEM作为稀释培养基，那必须在5min内混合稀释的DNA和Lipo2000。  

3）将DNA稀释液和Lipo2000稀释液混合（总体积100µl），轻柔混匀，室温静置20min， 形成DNA-Lipo2000复合物，这一混合物可能呈浑浊状态，但不会抑制转染效率。【注意】：DNA-Lipo2000复合物在室温下至少可稳定保存5h。

 

3.将上方制备好的DNA-Lipo2000复合物加入接种好的细胞中，将培养板轻轻地前后摇动，使复合物分散均匀。【注意】：如果要在无血清条件下转染，使用含血清的正常培养基进行细胞接种。再加入复合物前吸掉培养基，更换为500µl无血清培养基。

4. 37℃，5% CO2培养箱培养24-48h，直至能进行转基因表达分析，无需去掉复合物或更换培养基。然而，有必要在4-6 h后更换生长培养基，不会降低转染活性。

 

5.特殊说明

1）对于稳转细胞株：则在转染24h后，按照1:10或更高比例接种细胞到新鲜生长培养基。转染48h后加入筛选培养基。

2）对于悬浮细胞株：在细胞中加入DNA-Lipo2000复合物后，如需要可以4h后加入PMA和/或PHA。比如：对于Jurkat细胞，分别加入PHA-L（终浓度1µg/ml）和PMA（终浓度50ng/ml），可以提高CMV启动子活性和基因表达。对于K562细胞，只加入PMA（终浓度50ng/ml）足以提高启动子活性。

 

转染体系的放大或缩小

对于不同的细胞培养板，Lipo2000、DNA、细胞和培养基的使用量根据培养表面的不同按比例进行调整，具体参考表I。对于自动化、高通量体系，以96孔板形式制备更大的复合物体积。需要注意的是，需要进行快速的96孔板转染（细胞铺板和转染同时进行），直接在平板中制备复合物，然后将细胞悬液加入到复合物内，这样进一步减少了转染时间。此种改进步骤经过293-H，293-F，COS-7L和CHO细胞的试验，同传统方法相比活性稍低。

 

表I.不同细胞培养容器中转染时培养基、核酸及Lipo2000用量

培养容器 单孔表面积*           培养基用量 DNA转染 RNAi转染
铺板培养 基用量    稀释培养 基用量**        DNA          Lipo2000   siRNA Lipo200   
96-well 0.3 cm2 100µl 2 × 25 µl 0.2 µg 0.5 µl 5 pmol 0.25 µl
24-well 2 cm2 500µl 2 × 50 µl 0.8 µg 2.0 µl 20 pmol 1.0 µl
12-well 4 cm2 1 ml 2 × 100 µl 1.6 µg 4.0 µl 40 pmol 2.0 µl
6-well 10 cm2 2 ml 2 × 250 µl 4.0 µg 10 µl 100 pmol    5 µl
60-mm 20 cm2 5 ml 2 × 0.5 ml 8.0 µg 20 µl 200 pmol 10 µl
100-mm 60 cm2 15 ml 2 × 1.5 ml 24 µg 60 µl 600 pmol 30 µl

【*】：不同厂商提供的细胞培养容器表面积可能有所不同。

【**】：稀释DNA/RNA或Lipo2000所用的无血清培养基用量。

【注意】：该表用量仅供参考，具体用量请根据细胞类型、铺板密度等其他实验条件进行优化。

 

附表II  Lipo2000脂质体转染试剂用于不同细胞转染用量参考（以96孔板为例）

细胞类型                  培养基                  每孔细胞数                 DNA用量                          Lipo2000用量                  
293H DMEM 3×104 0.2 µg 0.5 µl
293FT DMEM 3×104 0.2 µg 0.5 µl
293E DMEM 3×104 0.2 µg 0.5 µl
293F DMEM 3×104 0.2 µg 0.5 µl
HeLa DMEM 2×104 0.3 µg 0.5 µl
HepG2 DMEM 3×104 0.5 µg 0.75 µl
A549 DMEM 2×104 0.3 µg 0.5 µl
COS7 DMEM 1.5×104 0.4 µg 0.5 µl
Caco2 MEM 3.5×104 0.3 µg 0.75 µl
BHK21 MEM 2×104 0.2 µg 0.5 µl
RAW264.7 DMEM 3×104 0.2 µg 0.5 µl
CHO-K1 IMDM+Pro 3×104 0.2 µg 0.5 µl
Sf9 SIM SF 5×104 0.4 µg 0.75 µl

 

                        

Lipo2000 Transfection Reagent 脂质体转染试剂

发表于由

描述

Lipo2000 Transfection Reagent 脂质体转染试剂

 

 

产品信息

产品名称 产品编号 规格 价格（元）         
Lipo2000 Transfection Reagent Lipo2000脂质体转染试剂             MX2211-0.15ML        0.15ml          350
Lipo2000 Transfection Reagent Lipo2000脂质体转染试剂 MX2211-0.75ML 0.75ml 950
Lipo2000 Transfection Reagent Lipo2000脂质体转染试剂 MX2211-1.50ML 1.50ml 1650
Lipo2000 Transfection Reagent Lipo2000脂质体转染试剂 MX2211-7.50ML 7.50ml 5750

 

产品描述

Lipo2000脂质体转染试剂（Lipo2000 Transfection Reagent）是一款多用途转染试剂，适用于核酸（DNA、RNA）的转染，能在绝大多数贴壁和悬浮细胞（哺乳动物细胞系）提供高效转染。独特的配方使其能直接加入培养基，血清的存在不会影响转染效率。转染后无需去除DNA- Lipo2000复合物或更换培养基，也可根据自身需求在转染4-6h后更换新鲜培养基。

 

本品以无菌液体形式提供，浓度为1mg/ml。其使用方法和Lipofectamine™ 2000 Transfection Reagent基本一致，并且经过对HEK293、HeLa等细胞的转染测试，转染效率和Lipofectamine™ 2000相当。

通常情况下，对于24孔板的DNA转染，每次用2μl左右，则1.5mlLipo2000约可转染750个孔；对于24孔板的siRNA转染，每次用1μl左右，则1.5mlLipo2000约可转染1500个孔；

保存与运输方法

保存：+4℃保存，1年有效。切勿冻存。

运输：冰袋运输。

 

注意事项

1）使用高纯的DNA或RNA有助于获得较高的转染效率，内毒素是影响转染效率高低的重要因素。

2）使用Lipo2000脂质体转染试剂需确保高细胞密度，建议转染时细胞密度达90-95%，有助于获得最高转染效率和表达水平，且能最小化高转染活性导致的细胞生长降低影响。可通过优化实验条件来降低细胞密度，但需注意不同实验间维持一个标准的接种步骤，因转染效率依赖于细胞培养密度。

 

3）转染过程中不要添加抗生素到培养基，否则会导致细胞死亡。

4）为了获得最佳的转染效率，制备转染复合物时要求用无血清培养基（比如Opti-MEM I）稀释DNA和转染试剂，因血清会影响复合物的形成。其他无血清培养基（比如DMEM）也能用来稀释DNA和转染试剂，但转染效率有可能会降低。另外，需要特别注意某些无血清配方会抑制Lipo2000介导的转染，比如CD293, SFMII, VP-SFM等。

 

5）初次使用制备复合物时，最好优化DNA（µg）和Lipo2000脂质体转染试剂（µl）的比例。DNA和Lipo2000的比例，绝大多数细胞系通常推荐1:2-1:3，比如：24孔板内接种0.5-2×105个细胞，使用0.8-1µg DNA和2-3µL Lipo2000。通过调整DNA/ Lipo2000优化转染效率很有必要。

 

6）Lipo2000脂质体转染试剂应该在4℃保存，不可冻存。使用后立即盖好盖子，避免长时间暴露在空气中，否则有可能导致脂质体氧化而影响转染效率。

7）Lipo2000脂质体转染试剂不能涡旋或离心，宜缓慢晃动混匀。

8）为了您的安全和健康，请穿实验服并戴一次性手套操作。

 

使用方法

转染步骤（以质粒DNA的24孔板为例）

【注意】：对大多数细胞来说，DNA（µg）和Lipo2000（µl）的比例为1:2~1:3。转染时高的细胞密度可以得到高的转染效率和表达水平，并能减少细胞毒性。

 

1. 细胞准备

1）贴壁细胞：转染前一天，用500 µl不含抗生素的培养基接种细胞，使之在转染时细胞达90-95%汇合（0.5~2×105 细胞/个孔，24孔板）。

2）悬浮细胞：转染当天，于准备DNA-Lipo2000复合物之前，用500µl不含抗生素的培养基接种4~8×105细胞即可。

 

2.按照以下体系配制DNA- Lipo2000脂质体转染试剂复合物：

1）对于每孔细胞，在EP管内分别加入50µl无血清培养基（比如Opti-MEM™ I Reduced Serum Medium）和0.8 µg DNA轻柔混匀，制成DNA稀释液。

2）对于每孔细胞，在EP管内分别加入50µl无血清培养基（比如Opti-MEM™ I Reduced Serum Medium）和2.0 µl Lipo2000（注意用前先混匀），轻柔混匀，制成Lipo2000稀释液，室温静置5min。【注意】：需要在30min内混合稀释的DNA和Lipo2000，更长的等待时间会降低活性。如果使用DMEM作为稀释培养基，那必须在5min内混合稀释的DNA和Lipo2000。  

3）将DNA稀释液和Lipo2000稀释液混合（总体积100µl），轻柔混匀，室温静置20min， 形成DNA-Lipo2000复合物，这一混合物可能呈浑浊状态，但不会抑制转染效率。【注意】：DNA-Lipo2000复合物在室温下至少可稳定保存5h。

 

3.将上方制备好的DNA-Lipo2000复合物加入接种好的细胞中，将培养板轻轻地前后摇动，使复合物分散均匀。【注意】：如果要在无血清条件下转染，使用含血清的正常培养基进行细胞接种。再加入复合物前吸掉培养基，更换为500µl无血清培养基。

4. 37℃，5% CO2培养箱培养24-48h，直至能进行转基因表达分析，无需去掉复合物或更换培养基。然而，有必要在4-6 h后更换生长培养基，不会降低转染活性。

 

5.特殊说明

1）对于稳转细胞株：则在转染24h后，按照1:10或更高比例接种细胞到新鲜生长培养基。转染48h后加入筛选培养基。

2）对于悬浮细胞株：在细胞中加入DNA-Lipo2000复合物后，如需要可以4h后加入PMA和/或PHA。比如：对于Jurkat细胞，分别加入PHA-L（终浓度1µg/ml）和PMA（终浓度50ng/ml），可以提高CMV启动子活性和基因表达。对于K562细胞，只加入PMA（终浓度50ng/ml）足以提高启动子活性。

 

转染体系的放大或缩小

对于不同的细胞培养板，Lipo2000、DNA、细胞和培养基的使用量根据培养表面的不同按比例进行调整，具体参考表I。对于自动化、高通量体系，以96孔板形式制备更大的复合物体积。需要注意的是，需要进行快速的96孔板转染（细胞铺板和转染同时进行），直接在平板中制备复合物，然后将细胞悬液加入到复合物内，这样进一步减少了转染时间。此种改进步骤经过293-H，293-F，COS-7L和CHO细胞的试验，同传统方法相比活性稍低。

 

表I.不同细胞培养容器中转染时培养基、核酸及Lipo2000用量

培养容器 单孔表面积*           培养基用量 DNA转染 RNAi转染
铺板培养 基用量    稀释培养 基用量**        DNA          Lipo2000   siRNA Lipo200   
96-well 0.3 cm2 100µl 2 × 25 µl 0.2 µg 0.5 µl 5 pmol 0.25 µl
24-well 2 cm2 500µl 2 × 50 µl 0.8 µg 2.0 µl 20 pmol 1.0 µl
12-well 4 cm2 1 ml 2 × 100 µl 1.6 µg 4.0 µl 40 pmol 2.0 µl
6-well 10 cm2 2 ml 2 × 250 µl 4.0 µg 10 µl 100 pmol    5 µl
60-mm 20 cm2 5 ml 2 × 0.5 ml 8.0 µg 20 µl 200 pmol 10 µl
100-mm 60 cm2 15 ml 2 × 1.5 ml 24 µg 60 µl 600 pmol 30 µl

【*】：不同厂商提供的细胞培养容器表面积可能有所不同。

【**】：稀释DNA/RNA或Lipo2000所用的无血清培养基用量。

【注意】：该表用量仅供参考，具体用量请根据细胞类型、铺板密度等其他实验条件进行优化。

 

附表II  Lipo2000脂质体转染试剂用于不同细胞转染用量参考（以96孔板为例）

细胞类型                  培养基                  每孔细胞数                 DNA用量                          Lipo2000用量                  
293H DMEM 3×104 0.2 µg 0.5 µl
293FT DMEM 3×104 0.2 µg 0.5 µl
293E DMEM 3×104 0.2 µg 0.5 µl
293F DMEM 3×104 0.2 µg 0.5 µl
HeLa DMEM 2×104 0.3 µg 0.5 µl
HepG2 DMEM 3×104 0.5 µg 0.75 µl
A549 DMEM 2×104 0.3 µg 0.5 µl
COS7 DMEM 1.5×104 0.4 µg 0.5 µl
Caco2 MEM 3.5×104 0.3 µg 0.75 µl
BHK21 MEM 2×104 0.2 µg 0.5 µl
RAW264.7 DMEM 3×104 0.2 µg 0.5 µl
CHO-K1 IMDM+Pro 3×104 0.2 µg 0.5 µl
Sf9 SIM SF 5×104 0.4 µg 0.75 µl

 

                        

Lipo2000 Transfection Reagent 脂质体转染试剂

发表于由

描述

Lipo2000 Transfection Reagent 脂质体转染试剂

 

 

产品信息

产品名称 产品编号 规格 价格（元）         
Lipo2000 Transfection Reagent Lipo2000脂质体转染试剂             MX2211-0.15ML        0.15ml          350
Lipo2000 Transfection Reagent Lipo2000脂质体转染试剂 MX2211-0.75ML 0.75ml 950
Lipo2000 Transfection Reagent Lipo2000脂质体转染试剂 MX2211-1.50ML 1.50ml 1650
Lipo2000 Transfection Reagent Lipo2000脂质体转染试剂 MX2211-7.50ML 7.50ml 5750

 

产品描述

Lipo2000脂质体转染试剂（Lipo2000 Transfection Reagent）是一款多用途转染试剂，适用于核酸（DNA、RNA）的转染，能在绝大多数贴壁和悬浮细胞（哺乳动物细胞系）提供高效转染。独特的配方使其能直接加入培养基，血清的存在不会影响转染效率。转染后无需去除DNA- Lipo2000复合物或更换培养基，也可根据自身需求在转染4-6h后更换新鲜培养基。

 

本品以无菌液体形式提供，浓度为1mg/ml。其使用方法和Lipofectamine™ 2000 Transfection Reagent基本一致，并且经过对HEK293、HeLa等细胞的转染测试，转染效率和Lipofectamine™ 2000相当。

通常情况下，对于24孔板的DNA转染，每次用2μl左右，则1.5mlLipo2000约可转染750个孔；对于24孔板的siRNA转染，每次用1μl左右，则1.5mlLipo2000约可转染1500个孔；

保存与运输方法

保存：+4℃保存，1年有效。切勿冻存。

运输：冰袋运输。

 

注意事项

1）使用高纯的DNA或RNA有助于获得较高的转染效率，内毒素是影响转染效率高低的重要因素。

2）使用Lipo2000脂质体转染试剂需确保高细胞密度，建议转染时细胞密度达90-95%，有助于获得最高转染效率和表达水平，且能最小化高转染活性导致的细胞生长降低影响。可通过优化实验条件来降低细胞密度，但需注意不同实验间维持一个标准的接种步骤，因转染效率依赖于细胞培养密度。

 

3）转染过程中不要添加抗生素到培养基，否则会导致细胞死亡。

4）为了获得最佳的转染效率，制备转染复合物时要求用无血清培养基（比如Opti-MEM I）稀释DNA和转染试剂，因血清会影响复合物的形成。其他无血清培养基（比如DMEM）也能用来稀释DNA和转染试剂，但转染效率有可能会降低。另外，需要特别注意某些无血清配方会抑制Lipo2000介导的转染，比如CD293, SFMII, VP-SFM等。

 

5）初次使用制备复合物时，最好优化DNA（µg）和Lipo2000脂质体转染试剂（µl）的比例。DNA和Lipo2000的比例，绝大多数细胞系通常推荐1:2-1:3，比如：24孔板内接种0.5-2×105个细胞，使用0.8-1µg DNA和2-3µL Lipo2000。通过调整DNA/ Lipo2000优化转染效率很有必要。

 

6）Lipo2000脂质体转染试剂应该在4℃保存，不可冻存。使用后立即盖好盖子，避免长时间暴露在空气中，否则有可能导致脂质体氧化而影响转染效率。

7）Lipo2000脂质体转染试剂不能涡旋或离心，宜缓慢晃动混匀。

8）为了您的安全和健康，请穿实验服并戴一次性手套操作。

 

使用方法

转染步骤（以质粒DNA的24孔板为例）

【注意】：对大多数细胞来说，DNA（µg）和Lipo2000（µl）的比例为1:2~1:3。转染时高的细胞密度可以得到高的转染效率和表达水平，并能减少细胞毒性。

 

1. 细胞准备

1）贴壁细胞：转染前一天，用500 µl不含抗生素的培养基接种细胞，使之在转染时细胞达90-95%汇合（0.5~2×105 细胞/个孔，24孔板）。

2）悬浮细胞：转染当天，于准备DNA-Lipo2000复合物之前，用500µl不含抗生素的培养基接种4~8×105细胞即可。

 

2.按照以下体系配制DNA- Lipo2000脂质体转染试剂复合物：

1）对于每孔细胞，在EP管内分别加入50µl无血清培养基（比如Opti-MEM™ I Reduced Serum Medium）和0.8 µg DNA轻柔混匀，制成DNA稀释液。

2）对于每孔细胞，在EP管内分别加入50µl无血清培养基（比如Opti-MEM™ I Reduced Serum Medium）和2.0 µl Lipo2000（注意用前先混匀），轻柔混匀，制成Lipo2000稀释液，室温静置5min。【注意】：需要在30min内混合稀释的DNA和Lipo2000，更长的等待时间会降低活性。如果使用DMEM作为稀释培养基，那必须在5min内混合稀释的DNA和Lipo2000。  

3）将DNA稀释液和Lipo2000稀释液混合（总体积100µl），轻柔混匀，室温静置20min， 形成DNA-Lipo2000复合物，这一混合物可能呈浑浊状态，但不会抑制转染效率。【注意】：DNA-Lipo2000复合物在室温下至少可稳定保存5h。

 

3.将上方制备好的DNA-Lipo2000复合物加入接种好的细胞中，将培养板轻轻地前后摇动，使复合物分散均匀。【注意】：如果要在无血清条件下转染，使用含血清的正常培养基进行细胞接种。再加入复合物前吸掉培养基，更换为500µl无血清培养基。

4. 37℃，5% CO2培养箱培养24-48h，直至能进行转基因表达分析，无需去掉复合物或更换培养基。然而，有必要在4-6 h后更换生长培养基，不会降低转染活性。

 

5.特殊说明

1）对于稳转细胞株：则在转染24h后，按照1:10或更高比例接种细胞到新鲜生长培养基。转染48h后加入筛选培养基。

2）对于悬浮细胞株：在细胞中加入DNA-Lipo2000复合物后，如需要可以4h后加入PMA和/或PHA。比如：对于Jurkat细胞，分别加入PHA-L（终浓度1µg/ml）和PMA（终浓度50ng/ml），可以提高CMV启动子活性和基因表达。对于K562细胞，只加入PMA（终浓度50ng/ml）足以提高启动子活性。

 

转染体系的放大或缩小

对于不同的细胞培养板，Lipo2000、DNA、细胞和培养基的使用量根据培养表面的不同按比例进行调整，具体参考表I。对于自动化、高通量体系，以96孔板形式制备更大的复合物体积。需要注意的是，需要进行快速的96孔板转染（细胞铺板和转染同时进行），直接在平板中制备复合物，然后将细胞悬液加入到复合物内，这样进一步减少了转染时间。此种改进步骤经过293-H，293-F，COS-7L和CHO细胞的试验，同传统方法相比活性稍低。

 

表I.不同细胞培养容器中转染时培养基、核酸及Lipo2000用量

培养容器 单孔表面积*           培养基用量 DNA转染 RNAi转染
铺板培养 基用量    稀释培养 基用量**        DNA          Lipo2000   siRNA Lipo200   
96-well 0.3 cm2 100µl 2 × 25 µl 0.2 µg 0.5 µl 5 pmol 0.25 µl
24-well 2 cm2 500µl 2 × 50 µl 0.8 µg 2.0 µl 20 pmol 1.0 µl
12-well 4 cm2 1 ml 2 × 100 µl 1.6 µg 4.0 µl 40 pmol 2.0 µl
6-well 10 cm2 2 ml 2 × 250 µl 4.0 µg 10 µl 100 pmol    5 µl
60-mm 20 cm2 5 ml 2 × 0.5 ml 8.0 µg 20 µl 200 pmol 10 µl
100-mm 60 cm2 15 ml 2 × 1.5 ml 24 µg 60 µl 600 pmol 30 µl

【*】：不同厂商提供的细胞培养容器表面积可能有所不同。

【**】：稀释DNA/RNA或Lipo2000所用的无血清培养基用量。

【注意】：该表用量仅供参考，具体用量请根据细胞类型、铺板密度等其他实验条件进行优化。

 

附表II  Lipo2000脂质体转染试剂用于不同细胞转染用量参考（以96孔板为例）

细胞类型                  培养基                  每孔细胞数                 DNA用量                          Lipo2000用量                  
293H DMEM 3×104 0.2 µg 0.5 µl
293FT DMEM 3×104 0.2 µg 0.5 µl
293E DMEM 3×104 0.2 µg 0.5 µl
293F DMEM 3×104 0.2 µg 0.5 µl
HeLa DMEM 2×104 0.3 µg 0.5 µl
HepG2 DMEM 3×104 0.5 µg 0.75 µl
A549 DMEM 2×104 0.3 µg 0.5 µl
COS7 DMEM 1.5×104 0.4 µg 0.5 µl
Caco2 MEM 3.5×104 0.3 µg 0.75 µl
BHK21 MEM 2×104 0.2 µg 0.5 µl
RAW264.7 DMEM 3×104 0.2 µg 0.5 µl
CHO-K1 IMDM+Pro 3×104 0.2 µg 0.5 µl
Sf9 SIM SF 5×104 0.4 µg 0.75 µl

 

                        

氯膦酸盐 SUV PEG 脂质体

发表于由

基本信息

产品名称 氯膦酸盐 SUV PEG 脂质体
英文名称 CLODRONATE SUV PEG LIPOSOMES
运输条件 冰袋运输

一般描述

产品描述

对照脂质体不含氯膦酸盐。它们仅含有 PBS，可用于对照实验，以查明注射氯膦酸盐脂质体后观察到的效果是否仅是由于巨噬细胞耗竭所致。

氯膦酸盐脂质体：人工制备的包裹氯膦酸盐的脂质囊泡的悬浮液。悬浮液中氯膦酸盐的浓度约为5毫克/毫升。氯膦酸盐以 CH2Na2Cl2O6P2·4 H2O 的形式封装在脂质体囊泡中。

到达后，脂质体应储存在 4 – 8 ºC（或 39 – 47 ºF）之间。脂质体悬浮液不应冷冻，也不应暴露在极端高温下。这会导致磷脂双分子层受到干扰，可能导致氯膦酸盐从脂质体中泄漏出来。

PBS 脂质体：包裹 PBS 水溶液的人工制备的脂质囊泡的悬浮液。这些不含氯膦酸盐，可用于对照实验。

荧光 DiI 脂质体：人工制备的脂质囊泡悬浮液，包裹着 PBS 水溶液，用荧光染料 DiI 标记。这些不含氯膦酸盐，可用于研究通过特定给药途径注射的脂质体是否能够到达待研究的巨噬细胞。


储存和使用说明

给药前，先让脂质体达到室温，轻轻摇晃或搅拌混悬液。脂质体往往会在一段时间后沉淀，导致小瓶中分布不均匀。当注射时间过长时，脂质体甚至可能在注射器中沉淀。如果使用同一个注射器注射多只动物，这可能会导致剂量不同。

不鼓励稀释悬浮液，但如有必要，请使用 PBS 或盐水。

我们建议客户在发货后 16 周内使用我们的脂质体制剂。强烈建议不要在到期日之后使用。在这段时间之后，污染的风险会增加，并且可能会发生轻微的功能丧失。


作用机制

巨噬细胞在免疫和非免疫防御机制中发挥重要作用。它们构成了抵御细菌、病毒和其他形式的微生物污染侵入脊椎动物体内的第一道防线。巨噬细胞是大细胞，几乎存在于所有身体组织中，它们可以有不同的形式和名称（例如，枯否细胞、肺泡巨噬细胞、小胶质细胞、破骨细胞、红髓巨噬细胞）。巨噬细胞“清除”，它们摄取和消化所有可能是潜在病原体的外来物质、微生物、癌细胞和细胞碎片。这个过程称为吞噬作用。巨噬细胞进一步调节许多非吞噬细胞的功能，主要是通过介导可溶性分子如细胞因子和趋化因子。它们参与先天免疫。

脂质体是人工制备的球体，由同心磷脂双层组成。当磷脂分散在水中时，亲水性头部将构成脂质体的两个外部部分，而疏水性脂肪酸基团将构成内部部分（见图 1）。水性隔室将双层隔开，亲水性分子可以溶解在其中，从而产生脂质体包裹的分子。氯膦酸盐（二氯亚甲基二膦酸盐或 Cl2MBP）是一种亲水分子，可以封装在磷脂双层中。游离的氯膦酸盐不易穿过细胞膜，并被肾系统从循环中迅速清除（即在几分钟内）。然而，当包裹在脂质体中时，氯膦酸盐脂质体会被巨噬细胞摄取而无法逃脱（见图 2）。磷脂双分子层被溶酶体磷脂酶消化，而氯膦酸盐不被消化并保留在巨噬细胞中。巨噬细胞摄入的磷脂双分子层和脂质体越多，氯膦酸盐就会在巨噬细胞内积累越多。超过一定的细胞内浓度，氯膦酸盐将通过启动其程序性细胞死亡，即细胞凋亡来消除巨噬细胞。

氯膦酸盐 SUV PEG 脂质体
图 1. 氯膦酸盐脂质体的示意图。囊泡由同心磷脂双层组成，由水性隔室隔开。双层由亲水和疏水基团组成。氯膦酸盐（此处以黑色方块表示）溶解在水溶液中并封装在脂质体中。
氯膦酸盐 SUV PEG 脂质体
图 2. 巨噬细胞摄取和消化氯膦酸盐脂质体的示意图。氯膦酸盐脂质体通过内吞作用被巨噬细胞摄取，然后与含有磷脂酶的溶酶体 (L) 融合（箭头）。磷脂酶破坏的磷脂双分子层越多，巨噬细胞内释放的氯膦酸盐（黑色方块）就越多。巨噬细胞最终通过凋亡被杀死。（N = 巨噬细胞核）。

因此，氯膦酸盐脂质体可用于通过消耗巨噬细胞来研究巨噬细胞的功能。例如，它们可以应用于各种自身免疫疾病、移植、神经系统疾病和基因治疗的模型。氯膦酸盐脂质体只有在可以达到的情况下才能消耗巨噬细胞。一些组织可以形成脂质体的屏障。通过选择氯膦酸盐脂质体的正确给药途径，可以耗尽特定器官或组织的巨噬细胞。
PBS 脂质体主要用于对照实验。然而，这些也可以通过饱和来阻止吞噬作用一段时间。因此，PBS 脂质体不代表正常健康、非阻塞、非抑制和非活化巨噬细胞的对照实验。当比较氯膦酸盐脂质体与 PBS 脂质体的效果时，效果可能低于预期。

DESCRIPTION

Control liposomes do not contain clodronate. Containing PBS only, they can be used for control experiments, in order to find out whether the effects observed after injection of clodronate liposomes are due to macrophage depletion exclusively.

Clodronate liposomes: a suspension of artificially prepared lipid vesicles encapsulating clodronate. The concentration of clodronate in the suspension is ca. 5 mg / mL. Clodronate is encapsulated in the liposomal vesicles in the form of CH2Na2Cl2O6P2·4 H2O.

PBS liposomes: a suspension of artificially prepared lipid vesicles encapsulating an aqueous PBS solution. These do not contain clodronate and can be used for control experiments.

Fluorescent DiI Liposomes: a suspension of artificially prepared lipid vesicles encapsulating an aqueous PBS solution, labelled with the fluorochrome DiI. These do not contain clodronate and can be used to investigate whether liposomes injected via a particular administration route are able to reach the macrophages to be studied.

STORAGE AND DIRECTIONS OF USE

Upon arrival, liposomes should be stored between 4 – 8 ºC (or 39 – 47 ºF). The liposomal suspensions should never be frozen, nor be exposed to extreme high temperatures. This can cause disturbances to the phospholipid bilayers, possibly leading to leakage of clodronate out of the liposome.

Before administration, let the liposomes reach room temperature first and gently shake or stir the suspension. Liposomes tend to precipitate after some time, causing an inhomogeneous distribution in the vial. When injection takes too much time, the liposomes may even precipitate in the syringe. If multiple animals are injected using the same syringe, this can cause differential dosing.

Dilution of the suspension is discouraged, but if necessary use PBS or saline.

We advise our customers to use our liposomal formulation within 16 weeks after shipment. Use after the expiry date has occurred is strongly discouraged. After this period the risk of contamination increases, and a slight loss of function could occur.

MECHANISM OF ACTION

Macrophages play an important role in immune and non-immune defence mechanisms. They form a first line of defence against bacterial, viral and other forms of microbiological contamination penetrating into the bodies of vertebrates. Macrophages are large cells, found in almost all bodily tissues where they can have varying forms and names (e.g. Kupffer cells, alveolar macrophages, microglia, osteoclasts, red pulp macrophages). Macrophages “scavenge”, they ingest and digest all foreign substances, microbes, cancer cells and cellular debris that might be potential pathogens. This process is called phagocytosis. Macrophages further regulate functions of many non-phagocytic cells, mainly through mediation of soluble molecules such as cytokines and chemokines. They are involved in innate immunity, adaptive immunity and can have (anti-) inflammatory effects.

Liposomes are artificially prepared spheres and consist of concentric phospholipid bilayers. When phospholipids are dispersed in water, the hydrophilic heads will make up both outer parts of the liposome, whereas the hydrophobic fatty acid groups will make up the inner part (see figure 1). Aqueous compartments separate the bilayers, and hydrophilic molecules can be dissolved in it, resulting in liposome-encapsulated molecules. Clodronate (dichloromethylene-bisphosphonate or Cl2MBP) is a hydrophilic molecule that can be encapsulated within phospholipid bilayers. Free clodronate does not easily cross cell membranes, and is rapidly cleared (i.e. within minutes) from circulation by the renal system. However, when entrapped in a liposome, the clodronate liposome is ingested by macrophages and cannot escape it (see figure 2). The phospholipid bilayers are digested by lysosomal phospholipases, whereas clodronate is not digested and remains in the macrophage. The more phospholipid bilayers and liposomes are ingested by the macrophage, the more clodronate will accumulate within the macrophage. Exceeding a certain intracellular concentration, clodronate will eliminate the macrophage by initiating its programmed cell death, i.e. apoptosis.

氯膦酸盐 SUV PEG 脂质体

Figure 1. Schematic representation of a clodronate liposome. The vesicle consists of concentric phospholipid bilayers, separated by aqueous compartments. The bilayers consist of a hydrophilic and hydrophobic group. Clodronate (represented here as black squares) are dissolved in the aqueous solution and encapsulated within the liposome.

氯膦酸盐 SUV PEG 脂质体

Figure 2. Schematic representation of clodronate liposome ingestion and digestion by a macrophage. Clodronate liposomes are ingested by the macrophages via endocytosis and then fused with the lysosomes (L) which contain phospholipases (arrowheads). The more phospholipid bilayers are disrupted by the phospholipases, the more clodronate (black squares) is released within the macrophage. Macrophages are ultimately killed through apoptosis. (N = nucleus of macrophage).

Thus, clodronate liposomes can be used to study macrophage functioning by depletion of macrophages. For instance, they can be applied in various models of autoimmune disease, transplantation, neurological disorders and gene therapy. Clodronate liposomes are only able to deplete macrophages if they can be reached. Some tissues can form barriers for the liposomes. By choosing the right administration route of clodronate liposomes, particular organs or tissues can be depleted of macrophages.

PBS liposomes are mostly used for control experiments. However, these too can block phagocytosis by saturation for certain periods of time. PBS liposomes thus do not represent a control experiment with normal healthy, non-blocked, non-suppressed and non-activated macrophages. When comparing effects of clodronate liposomes with PBS liposomes, the effects can therefore be less than expected.

ADMINISTRATION PROTOCOLS

Administration protocols should be chosen carefully and depend on several factors, for instance: the type of macrophage you intend to deplete (e.g. Kupffer cells, alveolar macrophages, microglia, osteoclasts, red pulp macrophages), the time in which you intend to maintain depletion (e.g. short or long term), the (animal) model, and other experimental factors. In vitro application of clodronate liposomes is possible, albeit that they are specifically suitable to study macrophages in vivo. Below you can see schematic representations of the tissues and macrophages that can be reached through different administration routes. Please note that not all tissues and routes are represented here.

Abbreviations: AL = lung alveoli, BM = bone marrow, BR = brain, BV = blood vessels / circulation, DA = draining area of lymph node, EY = eye, GU = gut / intestines, IV = intravenous delivery, KI = kidney, LI = liver, LN = lymph node, LU = lung, LV = lymph vessels, PE = peritoneal cavity, SP = spleen, SY = synovial cavity in joint, TE = testis, TR = trachea.

氯膦酸盐 SUV PEG 脂质体

In general, it is recommend to inject 100 µL of suspension / 10 grams of animal weight for intravenous injection. Raising the dosage considerably may lead to blockage of capillaries. Intravenous administration of clodronate liposomes will lead to maximum depletion of liver and spleen macrophages in ca. 24 hours. Dependent on the subset of macrophages they will remain depleted for ca. 5 days. After that time, new macrophages will replace the depleted ones: macrophage precursors, monocytes, that are formed in bone marrow and released in circulation will arrive at their destination and further differentiate into mature macrophages. Monocytes,macrophage precursors, can also be depleted. To prevent macrophage repopulation, multiple injections can be administered every 2-3 days to target monocytes. See for instance: Sunderkötter, C., Nikolic, T., Dillon, M. J., Van Rooijen, N., Stehling, M., Drevets, D. A., & Leenen, P. J. (2004). Subpopulations of mouse blood monocytes differ in maturation stage and inflammatory response. The Journal of Immunology, 172(7), 4410-4417.

氯膦酸盐 SUV PEG 脂质体

For intraperitoneal administration, the recommended injection dosis (i.e. 100 µL of suspension / 10 grams of animal weight) can be increased considerably. This route also depletes peritoneal macrophages, but will take longer to deplete macrophages in liver and spleen (ca. 3 days). Depletion is slower and more gradual, since the liposomes have to be carried from the peritoneal cavity to circulation by lymph flow via the thoracic duct, which is a passive form of transport.

氯膦酸盐 SUV PEG 脂质体

Local administration is often required to target macrophages that are difficult to reach, such as macrophages in testis or the phagocytic synovial lining cells.

氯膦酸盐 SUV PEG 脂质体

For depletion of alveolar macrophages, clodronate liposomes can be administered intranasally as well as intratracheally. The difference is that intranasal liposomes may be spoiled in the oesophagus if not administered properly, whereas intratracheal instillation will deliver all liposomes in the lung. Please note that these routes only target alveolar macrophages, and not interstitial macrophages. These can be targeted through depletion of blood monocytes in circulation. See for instance: Huang, L., Nazarova, E. V., Tan, S., Liu, Y., & Russell, D. G. (2018). Growth of Mycobacterium tuberculosis in vivo segregates with host macrophage metabolism and ontogeny. Journalof Experimental Medicine, 215(4), 1135-1152.

氯膦酸盐 SUV PEG 脂质体

For subcutaneous injection the maximum volume to be injected depends on the storage capacity of the injection site.

RESULTS

Sections of mouse liver, stained with monoclonal antibody F4/80 Normal liver with positive Kupffer cells.

氯膦酸盐 SUV PEG 脂质体

Liver of a mouse, 2 days after injection with clodronate-liposomes. Kupffer cells have been depleted

氯膦酸盐 SUV PEG 脂质体

Sections of mouse spleen stained for acid phosphatase Normal spleen with positive splenic macrophages

氯膦酸盐 SUV PEG 脂质体

Spleen of a mouse 2 days after injection with clodronate liposomes. Only few macrophages are left

氯膦酸盐 SUV PEG 脂质体

相关属性

储存温度 2-8°C储存
品牌 金畔生物

10417004 1041730410417006WHATMAN聚碳酸酯PC脂质体挤出滤膜10417104

发表于由

【简单介绍】

品牌 其他品牌

WHATMAN聚碳酸酯PC脂质体挤出滤膜10417104 10417004 10417304 10417006，Nuclepore径迹蚀刻聚碳酸酯膜由高品质的聚碳酸酯膜制成，具有明确的孔径，高流速和出色的化学抗性和耐热性。该膜表面光滑平整，并且呈现出非常低水平的可萃取物。

【详细说明】

WHATMAN聚碳酸酯PC脂质体挤出滤膜10417104 10417004 10417304 10417006

Nuclepore径迹蚀刻聚碳酸酯膜由高品质的聚碳酸酯膜制成，具有明确的孔径，高流速和出色的化学抗性和耐热性。该膜表面光滑平整，并且呈现出非常低水平的可萃取物。

 

10417101  NUC PC 13MM 0.4uM 100/PK（原货号110407 ）

10417001  NUC PC 13MM 0.2uM 100/PK（原货号110406 ）

10417301  NUC PC 13MM 0.8uM 100/PK（原货号110409 ）

10417401  NUC PC 13MM 5uM 100/PK（原货号110413 ）

10417501  NUC PC 13MM 8uM 100/PK（原货号110414 ）

10417006  NUC PC 25MM 0.2uM 100/PK（原货号110606 ）

10417106  NUC PC 25MM 0.4uM 100/PK（原货号110607 ）

10417206  NUC PC 25MM 0.6uM 100/PK（原货号110608 ）

10417306  NUC PC 25MM 0.8uM 100/PK（原货号110609 ）

10417406  NUC PC 25MM 5uM 100/PK（原货号110613 ）

10417506  NUC PC 25MM 8uM 100/PK（原货号110614 ）

10417309  NUC PC 37MM 0.8uM 100/PK（原货号110809 ）

10417012  NUC PC 47MM 0.2uM 100/PK（原货号111106 ）

10417112  NUC PC 47MM 0.4uM 100/PK（原货号111107）

10417212  NUC PC 47MM 0.6uM 100/PK（原货号111108 ）

10417312  NUC PC 47MM 0.8uM 100/PK（原货号111109 ）

10417412  NUC PC 47MM 5uM 100/PK（原货号111113 ）

10417512  NUC PC 47MM 8uM 100/PK（原货号111114 ）

10417014  NUC PC 50MM 0.2uM 100/PK（原货号111206 ）

10417114  NUC PC 50MM 0.4uM 100/PK（原货号111207 ）

10417414  NUC PC 50MM 5uM 100/PK（原货号111213 ）

10417018  NUC PC 90MM 0.2uM 25/PK（原货号111706 ）

10417118  NUC PC 90MM 0.4uM 25/PK（原货号111707 ）

10417116  NUC PC 81MM 0.4uM 25/PK（原货号111718 ）

10417031  NUC PC 142MM 0.2uM 25/PK（原货号112106 ）

10417131  NUC PC 142MM 0.4uM 25/PK（原货号112126 ）

10417331  NUC PC 142MM 0.8uM 25/PK（原货号112126 ）

10417051  NUC PC 293MM 0.2uM 25/PK（原货号112806 ）

10417039  NUC PC 293MM 0.2uM 100/PK（原货号112112 ）

10417004  NUC PC 19MM 0.2uM 100/PK（原货号800281）

10417104  NUC PC 19MM 0.4uM 100/PK（原货号800282）

10417304  NUC PC 19MM 0.8uM 100/PK（原货号800284）

10417606  CYL PC 25MM 0.2uM 100/PK（原货号7060-2502）

10417612  CYL PC 47MM 0.2uM 100/PK（原货号7060-4702）

WHATMAN聚碳酸酯PC脂质体挤出滤膜10417104 10417004 10417304 10417006

氯膦酸钠SUV-PEG脂质体与对照SUV-PEG脂质体

发表于由

基本信息

产品名称 氯膦酸钠SUV-PEG脂质体与对照SUV-PEG脂质体
英文名称 CLODRONATE SUV PEG LIPOSOMES & CONTROL SUV PEG LIPOSOMES
运输条件 冰袋运输

一般描述

产品组成

货号 CLODRONATE SUV PEG LIPOSOMES CONTROL SUV PEG LIPOSOMES
C419764-2×5ml C419508-5ml C419763-5ml
C419764-2×10ml C419508-10ml C419763-10ml
C419764-2×15ml C419508-15ml C419763-15ml
C419764-2×20ml C419508-20ml C419763-20ml
C419764-2×30ml C419508-30ml C419763-30ml
C419764-2×40ml C419508-40ml C419763-40ml
C419764-2×50ml C419508-50ml C419763-50ml


产品描述

氯膦酸钠SUV-PEG脂质体

人工球体由单个磷脂双层组成，包裹含有氯膦酸盐的PBS（磷酸盐缓冲盐水）水溶液。将含有脂质的特殊PEG添加到组合物中，允许血液中长时间循环，并减缓制剂的降解。脂质体悬浮液已挤出，直径为200纳米，储存时不会沉淀。悬浮液中氯膦酸盐的浓度约为5 mg/ml。

根据要求，可以通过额外的挤压步骤将尺寸减小到120-150nm。

对照SUV-PEG脂质体

对照脂质体不含氯膦酸盐。仅含PBS，可用于对照实验，以确定注射氯膦酸盐脂质体后观察到的效应是否完全是由于巨噬细胞耗竭所致。脂质体分散体是用一种特殊的含有脂质的PEG制备的，这种脂质可以在血液中长时间循环，减缓制剂的降解。该制剂已被挤出，直径为200nm，这使其成为氯膦酸钠SUV-PEG脂质体的首选对照品。

根据要求，可以通过额外的挤压步骤将尺寸减小到120-150nm。


储存和使用说明

给药前，先让脂质体达到室温，轻轻摇晃或搅拌混悬液。脂质体往往会在一段时间后沉淀，导致小瓶中分布不均匀。当注射时间过长时，脂质体甚至可能在注射器中沉淀。如果使用同一个注射器注射多只动物，这可能会导致剂量不同。

不鼓励稀释悬浮液，但如有必要，请使用 PBS 或盐水。

我们建议客户在发货后 16 周内使用我们的脂质体制剂。强烈建议不要在到期日之后使用。在这段时间之后，污染的风险会增加，并且可能会发生轻微的功能丧失。


作用机制

巨噬细胞在免疫和非免疫防御机制中发挥重要作用。它们构成了抵御细菌、病毒和其他形式的微生物污染侵入脊椎动物体内的第一道防线。巨噬细胞是大细胞，几乎存在于所有身体组织中，它们可以有不同的形式和名称（例如，枯否细胞、肺泡巨噬细胞、小胶质细胞、破骨细胞、红髓巨噬细胞）。巨噬细胞“清除”，它们摄取和消化所有可能是潜在病原体的外来物质、微生物、癌细胞和细胞碎片。这个过程称为吞噬作用。巨噬细胞进一步调节许多非吞噬细胞的功能，主要是通过介导可溶性分子如细胞因子和趋化因子。它们参与先天免疫。

脂质体是人工制备的球体，由同心磷脂双层组成。当磷脂分散在水中时，亲水性头部将构成脂质体的两个外部部分，而疏水性脂肪酸基团将构成内部部分（见图 1）。水性隔室将双层隔开，亲水性分子可以溶解在其中，从而产生脂质体包裹的分子。氯膦酸盐（二氯亚甲基二膦酸盐或 Cl2MBP）是一种亲水分子，可以封装在磷脂双层中。游离的氯膦酸盐不易穿过细胞膜，并被肾系统从循环中迅速清除（即在几分钟内）。然而，当包裹在脂质体中时，氯膦酸盐脂质体会被巨噬细胞摄取而无法逃脱（见图 2）。磷脂双分子层被溶酶体磷脂酶消化，而氯膦酸盐不被消化并保留在巨噬细胞中。巨噬细胞摄入的磷脂双分子层和脂质体越多，氯膦酸盐就会在巨噬细胞内积累越多。超过一定的细胞内浓度，氯膦酸盐将通过启动其程序性细胞死亡，即细胞凋亡来消除巨噬细胞。

氯膦酸钠SUV-PEG脂质体与对照SUV-PEG脂质体
图 1. 氯膦酸盐脂质体的示意图。囊泡由同心磷脂双层组成，由水性隔室隔开。双层由亲水和疏水基团组成。氯膦酸盐（此处以黑色方块表示）溶解在水溶液中并封装在脂质体中。
氯膦酸钠SUV-PEG脂质体与对照SUV-PEG脂质体
图 2. 巨噬细胞摄取和消化氯膦酸盐脂质体的示意图。氯膦酸盐脂质体通过内吞作用被巨噬细胞摄取，然后与含有磷脂酶的溶酶体 (L) 融合（箭头）。磷脂酶破坏的磷脂双分子层越多，巨噬细胞内释放的氯膦酸盐（黑色方块）就越多。巨噬细胞最终通过凋亡被杀死。（N = 巨噬细胞核）。

因此，氯膦酸盐脂质体可用于通过消耗巨噬细胞来研究巨噬细胞的功能。例如，它们可以应用于各种自身免疫疾病、移植、神经系统疾病和基因治疗的模型。氯膦酸盐脂质体只有在可以达到的情况下才能消耗巨噬细胞。一些组织可以形成脂质体的屏障。通过选择氯膦酸盐脂质体的正确给药途径，可以耗尽特定器官或组织的巨噬细胞。
PBS 脂质体主要用于对照实验。然而，这些也可以通过饱和来阻止吞噬作用一段时间。因此，PBS 脂质体不代表正常健康、非阻塞、非抑制和非活化巨噬细胞的对照实验。当比较氯膦酸盐脂质体与 PBS 脂质体的效果时，效果可能低于预期。

PRODUCT COMPOSITION

货号 CLODRONATE SUV PEG LIPOSOMES CONTROL SUV PEG LIPOSOMES
C419764-2×5ml C419508-5ml C419763-5ml
C419764-2×10ml C419508-10ml C419763-10ml
C419764-2×15ml C419508-15ml C419763-15ml
C419764-2×20ml C419508-20ml C419763-20ml
C419764-2×30ml C419508-30ml C419763-30ml
C419764-2×40ml C419508-40ml C419763-40ml
C419764-2×50ml C419508-50ml C419763-50ml


DESCRIPTION

Clodronate SUV PEG Liposomes

Artificial spheres consisting of a single phospholipid bilayer encapsulating an aqueous PBS (phosphate buffered saline) solution containing clodronate. A special PEG-containing lipid is added to the composition, which allows for long circulation in blood and slower degradation of the formulation. The Liposomal suspension has been extruded and sized to 200nm in diameter and will not tend to precipitate upon storage. The concentration of clodronate in the suspension is ca. 5 mg/ml.

Upon request, the size can be reduced to 120-150nm with an extra extrusion step.

Control SUV PEG Liposomes

Control liposomes do not contain clodronate. Containing PBS only, they can be used for control experiments, in order to find out whether the effects observed after injection of clodronate liposomes are due to macrophage depletion exclusively. The Liposomal dispersion has been prepared with a special PEG-containing lipid which allows for long circulation in blood and slower degradation of the formulation. This formulation has been extruded and sized to 200nm in diameter which makes it the preferred control for the Clodronate SUV PEG liposomes.

Upon request, the size can be reduced to 120-150nm with an extra extrusion step.


STORAGE AND DIRECTIONS OF USE

Upon arrival, liposomes should be stored between 4 – 8 ºC (or 39 – 47 ºF). The liposomal suspensions should never be frozen, nor be exposed to extreme high temperatures. This can cause disturbances to the phospholipid bilayers, possibly leading to leakage of clodronate out of the liposome.

Before administration, let the liposomes reach room temperature first and gently shake or stir the suspension. Liposomes tend to precipitate after some time, causing an inhomogeneous distribution in the vial. When injection takes too much time, the liposomes may even precipitate in the syringe. If multiple animals are injected using the same syringe, this can cause differential dosing.

Dilution of the suspension is discouraged, but if necessary use PBS or saline.

We advise our customers to use our liposomal formulation within 16 weeks after shipment. Use after the expiry date has occurred is strongly discouraged. After this period the risk of contamination increases, and a slight loss of function could occur.

MECHANISM OF ACTION

Macrophages play an important role in immune and non-immune defence mechanisms. They form a first line of defence against bacterial, viral and other forms of microbiological contamination penetrating into the bodies of vertebrates. Macrophages are large cells, found in almost all bodily tissues where they can have varying forms and names (e.g. Kupffer cells, alveolar macrophages, microglia, osteoclasts, red pulp macrophages). Macrophages “scavenge”, they ingest and digest all foreign substances, microbes, cancer cells and cellular debris that might be potential pathogens. This process is called phagocytosis. Macrophages further regulate functions of many non-phagocytic cells, mainly through mediation of soluble molecules such as cytokines and chemokines. They are involved in innate immunity, adaptive immunity and can have (anti-) inflammatory effects.

Liposomes are artificially prepared spheres and consist of concentric phospholipid bilayers. When phospholipids are dispersed in water, the hydrophilic heads will make up both outer parts of the liposome, whereas the hydrophobic fatty acid groups will make up the inner part (see figure 1). Aqueous compartments separate the bilayers, and hydrophilic molecules can be dissolved in it, resulting in liposome-encapsulated molecules. Clodronate (dichloromethylene-bisphosphonate or Cl2MBP) is a hydrophilic molecule that can be encapsulated within phospholipid bilayers. Free clodronate does not easily cross cell membranes, and is rapidly cleared (i.e. within minutes) from circulation by the renal system. However, when entrapped in a liposome, the clodronate liposome is ingested by macrophages and cannot escape it (see figure 2). The phospholipid bilayers are digested by lysosomal phospholipases, whereas clodronate is not digested and remains in the macrophage. The more phospholipid bilayers and liposomes are ingested by the macrophage, the more clodronate will accumulate within the macrophage. Exceeding a certain intracellular concentration, clodronate will eliminate the macrophage by initiating its programmed cell death, i.e. apoptosis.

氯膦酸钠SUV-PEG脂质体与对照SUV-PEG脂质体

Figure 1. Schematic representation of a clodronate liposome. The vesicle consists of concentric phospholipid bilayers, separated by aqueous compartments. The bilayers consist of a hydrophilic and hydrophobic group. Clodronate (represented here as black squares) are dissolved in the aqueous solution and encapsulated within the liposome.

氯膦酸钠SUV-PEG脂质体与对照SUV-PEG脂质体

Figure 2. Schematic representation of clodronate liposome ingestion and digestion by a macrophage. Clodronate liposomes are ingested by the macrophages via endocytosis and then fused with the lysosomes (L) which contain phospholipases (arrowheads). The more phospholipid bilayers are disrupted by the phospholipases, the more clodronate (black squares) is released within the macrophage. Macrophages are ultimately killed through apoptosis. (N = nucleus of macrophage).

Thus, clodronate liposomes can be used to study macrophage functioning by depletion of macrophages. For instance, they can be applied in various models of autoimmune disease, transplantation, neurological disorders and gene therapy. Clodronate liposomes are only able to deplete macrophages if they can be reached. Some tissues can form barriers for the liposomes. By choosing the right administration route of clodronate liposomes, particular organs or tissues can be depleted of macrophages.

PBS liposomes are mostly used for control experiments. However, these too can block phagocytosis by saturation for certain periods of time. PBS liposomes thus do not represent a control experiment with normal healthy, non-blocked, non-suppressed and non-activated macrophages. When comparing effects of clodronate liposomes with PBS liposomes, the effects can therefore be less than expected.

ADMINISTRATION PROTOCOLS

Administration protocols should be chosen carefully and depend on several factors, for instance: the type of macrophage you intend to deplete (e.g. Kupffer cells, alveolar macrophages, microglia, osteoclasts, red pulp macrophages), the time in which you intend to maintain depletion (e.g. short or long term), the (animal) model, and other experimental factors. In vitro application of clodronate liposomes is possible, albeit that they are specifically suitable to study macrophages in vivo. Below you can see schematic representations of the tissues and macrophages that can be reached through different administration routes. Please note that not all tissues and routes are represented here.

Abbreviations: AL = lung alveoli, BM = bone marrow, BR = brain, BV = blood vessels / circulation, DA = draining area of lymph node, EY = eye, GU = gut / intestines, IV = intravenous delivery, KI = kidney, LI = liver, LN = lymph node, LU = lung, LV = lymph vessels, PE = peritoneal cavity, SP = spleen, SY = synovial cavity in joint, TE = testis, TR = trachea.

氯膦酸钠SUV-PEG脂质体与对照SUV-PEG脂质体

In general, it is recommend to inject 100 µL of suspension / 10 grams of animal weight for intravenous injection. Raising the dosage considerably may lead to blockage of capillaries. Intravenous administration of clodronate liposomes will lead to maximum depletion of liver and spleen macrophages in ca. 24 hours. Dependent on the subset of macrophages they will remain depleted for ca. 5 days. After that time, new macrophages will replace the depleted ones: macrophage precursors, monocytes, that are formed in bone marrow and released in circulation will arrive at their destination and further differentiate into mature macrophages. Monocytes,macrophage precursors, can also be depleted. To prevent macrophage repopulation, multiple injections can be administered every 2-3 days to target monocytes. See for instance: Sunderkötter, C., Nikolic, T., Dillon, M. J., Van Rooijen, N., Stehling, M., Drevets, D. A., & Leenen, P. J. (2004). Subpopulations of mouse blood monocytes differ in maturation stage and inflammatory response. The Journal of Immunology, 172(7), 4410-4417.

氯膦酸钠SUV-PEG脂质体与对照SUV-PEG脂质体

For intraperitoneal administration, the recommended injection dosis (i.e. 100 µL of suspension / 10 grams of animal weight) can be increased considerably. This route also depletes peritoneal macrophages, but will take longer to deplete macrophages in liver and spleen (ca. 3 days). Depletion is slower and more gradual, since the liposomes have to be carried from the peritoneal cavity to circulation by lymph flow via the thoracic duct, which is a passive form of transport.

氯膦酸钠SUV-PEG脂质体与对照SUV-PEG脂质体

Local administration is often required to target macrophages that are difficult to reach, such as macrophages in testis or the phagocytic synovial lining cells.

氯膦酸钠SUV-PEG脂质体与对照SUV-PEG脂质体

For depletion of alveolar macrophages, clodronate liposomes can be administered intranasally as well as intratracheally. The difference is that intranasal liposomes may be spoiled in the oesophagus if not administered properly, whereas intratracheal instillation will deliver all liposomes in the lung. Please note that these routes only target alveolar macrophages, and not interstitial macrophages. These can be targeted through depletion of blood monocytes in circulation. See for instance: Huang, L., Nazarova, E. V., Tan, S., Liu, Y., & Russell, D. G. (2018). Growth of Mycobacterium tuberculosis in vivo segregates with host macrophage metabolism and ontogeny. Journalof Experimental Medicine, 215(4), 1135-1152.

氯膦酸钠SUV-PEG脂质体与对照SUV-PEG脂质体

For subcutaneous injection the maximum volume to be injected depends on the storage capacity of the injection site.

RESULTS

Sections of mouse liver, stained with monoclonal antibody F4/80 Normal liver with positive Kupffer cells.

氯膦酸钠SUV-PEG脂质体与对照SUV-PEG脂质体

Liver of a mouse, 2 days after injection with clodronate-liposomes. Kupffer cells have been depleted

氯膦酸钠SUV-PEG脂质体与对照SUV-PEG脂质体

Sections of mouse spleen stained for acid phosphatase Normal spleen with positive splenic macrophages

氯膦酸钠SUV-PEG脂质体与对照SUV-PEG脂质体

Spleen of a mouse 2 days after injection with clodronate liposomes. Only few macrophages are left

氯膦酸钠SUV-PEG脂质体与对照SUV-PEG脂质体

相关属性

储存温度 2-8°C储存
品牌 金畔生物

对照SUV-PEG脂质体

发表于由

基本信息

产品名称 对照SUV-PEG脂质体
英文名称 CONTROL SUV PEG LIPOSOMES
运输条件 冰袋运输

一般描述

产品描述

对照SUV PEG脂质体：

对照脂质体不含氯膦酸盐。仅含PBS，可用于对照实验，以确定注射氯膦酸盐脂质体后观察到的效应是否完全是由于巨噬细胞耗竭所致。脂质体分散体是用一种特殊的含有脂质的PEG制备的，这种脂质可以在血液中长时间循环，减缓制剂的降解。该制剂已被挤出，直径为200nm，这使其成为氯膦酸钠SUV-PEG脂质体的首选对照品。

根据要求，可以通过额外的挤压步骤将尺寸减小到120-150nm。


储存和使用说明

给药前，先让脂质体达到室温，轻轻摇晃或搅拌混悬液。脂质体往往会在一段时间后沉淀，导致小瓶中分布不均匀。当注射时间过长时，脂质体甚至可能在注射器中沉淀。如果使用同一个注射器注射多只动物，这可能会导致剂量不同。

不鼓励稀释悬浮液，但如有必要，请使用 PBS 或盐水。

我们建议客户在发货后 16 周内使用我们的脂质体制剂。强烈建议不要在到期日之后使用。在这段时间之后，污染的风险会增加，并且可能会发生轻微的功能丧失。


作用机制

巨噬细胞在免疫和非免疫防御机制中发挥重要作用。它们构成了抵御细菌、病毒和其他形式的微生物污染侵入脊椎动物体内的第一道防线。巨噬细胞是大细胞，几乎存在于所有身体组织中，它们可以有不同的形式和名称（例如，枯否细胞、肺泡巨噬细胞、小胶质细胞、破骨细胞、红髓巨噬细胞）。巨噬细胞“清除”，它们摄取和消化所有可能是潜在病原体的外来物质、微生物、癌细胞和细胞碎片。这个过程称为吞噬作用。巨噬细胞进一步调节许多非吞噬细胞的功能，主要是通过介导可溶性分子如细胞因子和趋化因子。它们参与先天免疫。

脂质体是人工制备的球体，由同心磷脂双层组成。当磷脂分散在水中时，亲水性头部将构成脂质体的两个外部部分，而疏水性脂肪酸基团将构成内部部分（见图 1）。水性隔室将双层隔开，亲水性分子可以溶解在其中，从而产生脂质体包裹的分子。氯膦酸盐（二氯亚甲基二膦酸盐或 Cl2MBP）是一种亲水分子，可以封装在磷脂双层中。游离的氯膦酸盐不易穿过细胞膜，并被肾系统从循环中迅速清除（即在几分钟内）。然而，当包裹在脂质体中时，氯膦酸盐脂质体会被巨噬细胞摄取而无法逃脱（见图 2）。磷脂双分子层被溶酶体磷脂酶消化，而氯膦酸盐不被消化并保留在巨噬细胞中。巨噬细胞摄入的磷脂双分子层和脂质体越多，氯膦酸盐就会在巨噬细胞内积累越多。超过一定的细胞内浓度，氯膦酸盐将通过启动其程序性细胞死亡，即细胞凋亡来消除巨噬细胞。

对照SUV-PEG脂质体
图 1. 氯膦酸盐脂质体的示意图。囊泡由同心磷脂双层组成，由水性隔室隔开。双层由亲水和疏水基团组成。氯膦酸盐（此处以黑色方块表示）溶解在水溶液中并封装在脂质体中。
对照SUV-PEG脂质体
图 2. 巨噬细胞摄取和消化氯膦酸盐脂质体的示意图。氯膦酸盐脂质体通过内吞作用被巨噬细胞摄取，然后与含有磷脂酶的溶酶体 (L) 融合（箭头）。磷脂酶破坏的磷脂双分子层越多，巨噬细胞内释放的氯膦酸盐（黑色方块）就越多。巨噬细胞最终通过凋亡被杀死。（N = 巨噬细胞核）。

因此，氯膦酸盐脂质体可用于通过消耗巨噬细胞来研究巨噬细胞的功能。例如，它们可以应用于各种自身免疫疾病、移植、神经系统疾病和基因治疗的模型。氯膦酸盐脂质体只有在可以达到的情况下才能消耗巨噬细胞。一些组织可以形成脂质体的屏障。通过选择氯膦酸盐脂质体的正确给药途径，可以耗尽特定器官或组织的巨噬细胞。
PBS 脂质体主要用于对照实验。然而，这些也可以通过饱和来阻止吞噬作用一段时间。因此，PBS 脂质体不代表正常健康、非阻塞、非抑制和非活化巨噬细胞的对照实验。当比较氯膦酸盐脂质体与 PBS 脂质体的效果时，效果可能低于预期。

DESCRIPTION

CONTROL SUV PEG LIPOSOMES:

Control liposomes do not contain clodronate. Containing PBS only, they can be used for control experiments, in order to find out whether the effects observed after injection of clodronate liposomes are due to macrophage depletion exclusively. The Liposomal dispersion has been prepared with a special PEG-containing lipid which allows for long circulation in blood and slower degradation of the formulation. This formulation has been extruded and sized to 200nm in diameter which makes it the preferred control for the Clodronate SUV PEG liposomes.

Upon request, the size can be reduced to 120-150nm with an extra extrusion step.

STORAGE AND DIRECTIONS OF USE

Upon arrival, liposomes should be stored between 4 – 8 ºC (or 39 – 47 ºF). The liposomal suspensions should never be frozen, nor be exposed to extreme high temperatures. This can cause disturbances to the phospholipid bilayers, possibly leading to leakage of clodronate out of the liposome.

Before administration, let the liposomes reach room temperature first and gently shake or stir the suspension. Liposomes tend to precipitate after some time, causing an inhomogeneous distribution in the vial. When injection takes too much time, the liposomes may even precipitate in the syringe. If multiple animals are injected using the same syringe, this can cause differential dosing.

Dilution of the suspension is discouraged, but if necessary use PBS or saline.

We advise our customers to use our liposomal formulation within 16 weeks after shipment. Use after the expiry date has occurred is strongly discouraged. After this period the risk of contamination increases, and a slight loss of function could occur.

MECHANISM OF ACTION

Macrophages play an important role in immune and non-immune defence mechanisms. They form a first line of defence against bacterial, viral and other forms of microbiological contamination penetrating into the bodies of vertebrates. Macrophages are large cells, found in almost all bodily tissues where they can have varying forms and names (e.g. Kupffer cells, alveolar macrophages, microglia, osteoclasts, red pulp macrophages). Macrophages “scavenge”, they ingest and digest all foreign substances, microbes, cancer cells and cellular debris that might be potential pathogens. This process is called phagocytosis. Macrophages further regulate functions of many non-phagocytic cells, mainly through mediation of soluble molecules such as cytokines and chemokines. They are involved in innate immunity, adaptive immunity and can have (anti-) inflammatory effects.

Liposomes are artificially prepared spheres and consist of concentric phospholipid bilayers. When phospholipids are dispersed in water, the hydrophilic heads will make up both outer parts of the liposome, whereas the hydrophobic fatty acid groups will make up the inner part (see figure 1). Aqueous compartments separate the bilayers, and hydrophilic molecules can be dissolved in it, resulting in liposome-encapsulated molecules. Clodronate (dichloromethylene-bisphosphonate or Cl2MBP) is a hydrophilic molecule that can be encapsulated within phospholipid bilayers. Free clodronate does not easily cross cell membranes, and is rapidly cleared (i.e. within minutes) from circulation by the renal system. However, when entrapped in a liposome, the clodronate liposome is ingested by macrophages and cannot escape it (see figure 2). The phospholipid bilayers are digested by lysosomal phospholipases, whereas clodronate is not digested and remains in the macrophage. The more phospholipid bilayers and liposomes are ingested by the macrophage, the more clodronate will accumulate within the macrophage. Exceeding a certain intracellular concentration, clodronate will eliminate the macrophage by initiating its programmed cell death, i.e. apoptosis.

对照SUV-PEG脂质体

Figure 1. Schematic representation of a clodronate liposome. The vesicle consists of concentric phospholipid bilayers, separated by aqueous compartments. The bilayers consist of a hydrophilic and hydrophobic group. Clodronate (represented here as black squares) are dissolved in the aqueous solution and encapsulated within the liposome.

对照SUV-PEG脂质体

Figure 2. Schematic representation of clodronate liposome ingestion and digestion by a macrophage. Clodronate liposomes are ingested by the macrophages via endocytosis and then fused with the lysosomes (L) which contain phospholipases (arrowheads). The more phospholipid bilayers are disrupted by the phospholipases, the more clodronate (black squares) is released within the macrophage. Macrophages are ultimately killed through apoptosis. (N = nucleus of macrophage).

Thus, clodronate liposomes can be used to study macrophage functioning by depletion of macrophages. For instance, they can be applied in various models of autoimmune disease, transplantation, neurological disorders and gene therapy. Clodronate liposomes are only able to deplete macrophages if they can be reached. Some tissues can form barriers for the liposomes. By choosing the right administration route of clodronate liposomes, particular organs or tissues can be depleted of macrophages.

PBS liposomes are mostly used for control experiments. However, these too can block phagocytosis by saturation for certain periods of time. PBS liposomes thus do not represent a control experiment with normal healthy, non-blocked, non-suppressed and non-activated macrophages. When comparing effects of clodronate liposomes with PBS liposomes, the effects can therefore be less than expected.

ADMINISTRATION PROTOCOLS

Administration protocols should be chosen carefully and depend on several factors, for instance: the type of macrophage you intend to deplete (e.g. Kupffer cells, alveolar macrophages, microglia, osteoclasts, red pulp macrophages), the time in which you intend to maintain depletion (e.g. short or long term), the (animal) model, and other experimental factors. In vitro application of clodronate liposomes is possible, albeit that they are specifically suitable to study macrophages in vivo. Below you can see schematic representations of the tissues and macrophages that can be reached through different administration routes. Please note that not all tissues and routes are represented here.

Abbreviations: AL = lung alveoli, BM = bone marrow, BR = brain, BV = blood vessels / circulation, DA = draining area of lymph node, EY = eye, GU = gut / intestines, IV = intravenous delivery, KI = kidney, LI = liver, LN = lymph node, LU = lung, LV = lymph vessels, PE = peritoneal cavity, SP = spleen, SY = synovial cavity in joint, TE = testis, TR = trachea.

对照SUV-PEG脂质体

In general, it is recommend to inject 100 µL of suspension / 10 grams of animal weight for intravenous injection. Raising the dosage considerably may lead to blockage of capillaries. Intravenous administration of clodronate liposomes will lead to maximum depletion of liver and spleen macrophages in ca. 24 hours. Dependent on the subset of macrophages they will remain depleted for ca. 5 days. After that time, new macrophages will replace the depleted ones: macrophage precursors, monocytes, that are formed in bone marrow and released in circulation will arrive at their destination and further differentiate into mature macrophages. Monocytes,macrophage precursors, can also be depleted. To prevent macrophage repopulation, multiple injections can be administered every 2-3 days to target monocytes. See for instance: Sunderkötter, C., Nikolic, T., Dillon, M. J., Van Rooijen, N., Stehling, M., Drevets, D. A., & Leenen, P. J. (2004). Subpopulations of mouse blood monocytes differ in maturation stage and inflammatory response. The Journal of Immunology, 172(7), 4410-4417.

对照SUV-PEG脂质体

For intraperitoneal administration, the recommended injection dosis (i.e. 100 µL of suspension / 10 grams of animal weight) can be increased considerably. This route also depletes peritoneal macrophages, but will take longer to deplete macrophages in liver and spleen (ca. 3 days). Depletion is slower and more gradual, since the liposomes have to be carried from the peritoneal cavity to circulation by lymph flow via the thoracic duct, which is a passive form of transport.

对照SUV-PEG脂质体

Local administration is often required to target macrophages that are difficult to reach, such as macrophages in testis or the phagocytic synovial lining cells.

对照SUV-PEG脂质体

For depletion of alveolar macrophages, clodronate liposomes can be administered intranasally as well as intratracheally. The difference is that intranasal liposomes may be spoiled in the oesophagus if not administered properly, whereas intratracheal instillation will deliver all liposomes in the lung. Please note that these routes only target alveolar macrophages, and not interstitial macrophages. These can be targeted through depletion of blood monocytes in circulation. See for instance: Huang, L., Nazarova, E. V., Tan, S., Liu, Y., & Russell, D. G. (2018). Growth of Mycobacterium tuberculosis in vivo segregates with host macrophage metabolism and ontogeny. Journalof Experimental Medicine, 215(4), 1135-1152.

对照SUV-PEG脂质体

For subcutaneous injection the maximum volume to be injected depends on the storage capacity of the injection site.

RESULTS

Sections of mouse liver, stained with monoclonal antibody F4/80 Normal liver with positive Kupffer cells.

对照SUV-PEG脂质体

Liver of a mouse, 2 days after injection with clodronate-liposomes. Kupffer cells have been depleted

对照SUV-PEG脂质体

Sections of mouse spleen stained for acid phosphatase Normal spleen with positive splenic macrophages

对照SUV-PEG脂质体

Spleen of a mouse 2 days after injection with clodronate liposomes. Only few macrophages are left

对照SUV-PEG脂质体

相关属性

储存温度 2-8°C储存
品牌 金畔生物

Whatman脂质体挤出聚碳酸酯膜0.2um19mm

发表于由
产品简介

Whatman脂质体挤出聚碳酸酯膜0.2um19mm，原货号800281，挤压制备盐酸洛拉曲克脂质体，制备肿瘤药物脂质体，用于细胞培养、趋药性和细胞毒性分析。

详情介绍

Whatman脂质体挤出聚碳酸酯膜0.2um19mm

径迹蚀刻膜（核孔膜）由Whatman先进技术生产，包括NulcleporeTM和CycleporeTM聚碳酸酯膜、趋药性膜、黑色核孔膜和细胞培养膜等。径迹蚀刻膜（Track-Etched Membrane）圆柱形孔贯穿膜基体，能够精确截留，膜表面光滑平整，方便显微镜表面观察。径迹蚀刻膜由纯聚合物制成，亲水性好，还提供疏水性膜。化学洁净度好，不含污染物或杂质，皮重zui轻、吸水量zui小和极低的非特异蛋白吸附，它耐受盐酸和硝酸、醇、醚、环己烷等，广泛化学兼容性非常适于检测许多腐蚀性及有机液体中的颗粒物。

Whatman脂质体挤出聚碳酸酯膜0.2um19mm

特性和优势

  • 不粘附染色剂，光学反差大，有利于用显微镜观察；
  • 真正的表面截留，更易进行样品测定并缩短分析时间
  • 供应完全透明的径迹蚀刻膜
  • 不吸潮，滤液吸附zui小，皮重极小
  • 没有颗粒脱落，确保滤液洁净度，生物学惰性

典型应用

  • 空气污染监测

化学及放射元素/颗粒分析（灰尘、石棉、花粉和气溶胶）

  • 制备肿瘤药物脂质体

阿霉素热敏、shRNA-survivin、姜黄素、拉曲克长、盐酸洛拉脂质体等挤压制备

  • 分析方法

重量法、比色法、吸光值、发射光谱、XRF和IR

  • 水分析

吸附有机卤素(AOX)、微生物直接计数、海洋生物学和溶解磷酸盐、硝酸盐和氨盐分析

  • 石油/燃料检测

检测汽油、柴油和残渣油的不溶性颗粒物

  • 血液过滤和细胞分析

红细胞变形性、去除白血球、红细胞过滤和血浆提取法、趋药性、细胞学和细胞培养

  • 普通过滤实验

去除颗粒物和细菌、冷灭菌过滤、横向流过滤、HPLC样品制备、流动相过滤

  • 显微镜观察

电子显微镜、落射荧光显微镜、光学显微镜

  • 微生物分析

微生物总数直接计数、采集/收集、浓缩、分级（酵母菌、霉菌、贾滴虫、军团病杆菌、大肠杆菌和齿状微丝蚴）

  • 核酸研究

碱洗提和DNA-片段分级分离

  • 海洋学研究

透明聚碳酸酯膜极适合研究浮游生物。超薄透明膜柔韧性和强度好，浮游生物被过滤后可直接装入载玻片观察

  • 医疗领域

生物传感器―电化学探针和生物试剂可控扩散的隔膜
诊断―微流体芯片、样品制备、血液分离和捕获胶乳微粒
细胞生物学―用于细胞培养、趋药性和细胞毒性分析，例如直接染色、同位素标记和基于荧光的分析皮下药物释放-作为截留治疗药物的惰性载体

脂质体挤出滤膜

发表于由

【简单介绍】

品牌 其他品牌

脂质体挤出滤膜，Nuclepore径迹蚀刻聚碳酸酯膜由高品质的聚碳酸酯膜制成，具有明确的孔径，高流速和出色的化学抗性和耐热性。

【详细说明】

脂质体挤出滤膜 在脂质体剂型研发和生产过程中，如何去除产品中的颗粒和沉淀，减小脂质体及乳剂粒径以便于无菌过滤，使产品粒径均一化是确保药品安全很重要的工艺开发步骤。减小脂质体粒径方法很多，近年来采用膜挤出的新技术效果优良，同时易于实现工业放大，还原实验室工艺开发结果。脂质体的*终粒径大小与摸索的挤出压力及选择PC滤膜的孔径有关，PC滤膜的孔径大小是主要因素，膜的直径大小选择主要看处理量。

滤膜水处理是固液分离技术，它是以膜孔把水滤过，将水中杂质截留，而没有化学变化，处理简易的技术，但因膜孔非常细小，相应的存在某些技术问题。在给水也有用生物膜处理原水的方法，但它与过滤膜分离技术不同。用作膜分离的叫做membrane，用作生物膜处理的膜叫做film.

脂质体挤出滤膜

10417101  NUC PC 13MM 0.4uM 100/PK（原货号110407 ）

10417001  NUC PC 13MM 0.2uM 100/PK（原货号110406 ）

10417301  NUC PC 13MM 0.8uM 100/PK（原货号110409 ）

10417401  NUC PC 13MM 5uM 100/PK（原货号110413 ）

10417501  NUC PC 13MM 8uM 100/PK（原货号110414 ）

10417006  NUC PC 25MM 0.2uM 100/PK（原货号110606 ）

10417106  NUC PC 25MM 0.4uM 100/PK（原货号110607 ）