How to separate the macrophage from the bottom of the plate so that the cell does not lyse?
Hello Nooshin Ghadiri
You should try to culture macrophages on low adhesion plastic and harvest them by placing the plate in the refrigerator at 4 degree C for 15 to 20 mins. This will help to loosen the cells from the substratum, and then later you may remove most of them off from the bottom of the plate using the pipette.
Alternatively, you could use ice cold sterile PBS (w/o Ca2+ and Mg2+) and incubate for 20-30 minutes at 4 degree C. You may later give the plate a quick wash with sterile PBS (w/o Ca2+ and Mg2+) before lifting the cells.
Best.
I can recommend UpCell Plates from Nunc. They detach adherent cells non-enzymatically via temperature differences. All other methods such as PBS+EDTA, ice cold PBS or Accutase on regular cell culture plates will leave you with residual cell attachted to the plastic or compromised viability of the cells.
Good luck!
For adherent macrophage culture, you have only to wash cells three times with ice-cold PBS. The free particles will be removed in this washs. To remove the macrofages from culture dish, you can use RPMI 1640 medium or the medium that you are using adding trypsin- ethylenediaminetetraacetic acid (EDTA) solution to detach the macrophages .After that, you can analysed by microscopy or by Facs.
I recommend culturing your cells in non-treated plastic (like petri dish for bacteria culture but sterile). For detaching them place the plate on ice, cold PBS for 10 mins, change for PBS+EDTA 5mM 5 mins, neutralize the reaction with complete media V/V, and pipette up and down.
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