Dear Dr. Vahinipriya Manoharan

Macrophages become strongly adherent to the culture dish, making it difficult to detach them. It would not be proper to leave them in trypsin for 30 minutes. Instead, there are other alternatives that I have listed below which you could incorporate in your protocol.

1. Macrophages can be lifted by incubating them in cold PBS (without Ca2+ and Mg2+) and 5 mM EDTA for 45 min at 4 °C.

Or

2. Incubate the cells in ice-cold PBS (50% of media volume) for 5 min. Detach the cells from the plate with gentle scraping or repeated pipetting.

Or

3. Make use of temperature-responsive culture dish coated with poly-(N-isopropylacrylamide). On lowering to room temperature, the culture surfaces change from hydrophobic to hydrophilic, and this weakens the cells' attachment to the dish. For more information on temperature-responsive culture dish coated with poly-(N-isopropylacrylamide), you may want to refer to the papers attached below.

Article A novel recovery system for cultured cells using plasma-trea...

Article Poly( N -isopropylacrylamide)-Based Thermoresponsive Surface...

Good Luck!

Regards,

Malcolm Nobre

Thank you so much

Dear Vahinipriya Manoharan ,

please take a look here:

https://www.researchgate.net/post/Is_passaging_of_PBMC-derived_macrophages_feasible

Best wishes Soenke

I wash the cells twice with PBS, then add trypsin and incubate for 15 minutes. After that, I pipette the trypsin over the attached cells about 20 times, and then add 10% medium to harvest the M2 cells. This works for me