I am doing BMDMs and I can not detach macrophages , they are so sticky and trypsin is not enough to detach them.
would scrapper works or will it destroy the cells ?
Hello Nora Kh
Macrophages adhere strongly to tissue culture-treated vessels. You may use EDTA in combination with cold shock at 4°C to enhance the dissociation effect. Use 5mM EDTA in PBS (without Ca2+ and Mg2+) and incubate at 4°C (refrigerator) for 15-20 mins followed by good pipetting to detach the macrophages.
Alternatively, if you are using trypsin, you may allow the culture vessel to incubate with trypsin for 10–15 min at 37°C and pipet vigorously at points all around the circumference of the vessel to lift the cells. If cells are still attached, repeat the pipetting process with PBS (without Ca2+ and Mg2+) to collect the remaining cells. I would not recommend the use of cell scraper as this can damage the macrophages.
Best.
You can use citrate saline (135 mM potassium chloride plus 15 mM sodium citrate in H2O, autoclaved), add to the plate and incubate them 5 min at 37 °C. You can gently tap the plate to help detach. Give a wash with PBS before plating.
You might also give Accutase a try.
http://www.accutase.com/accutase.html
http://www.accutase.com/macrophage-passaging-protocol.html
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