macrophages culture, please help - (Jun/22/2006 )
Maybe somebody could help me with my question:
i wonder why people use human serum to cover plastic before they put monocytes on it?
may i use FBS instead HS?
i tried to isolate monocytes from PBMCs by adherance protocol. So i allowed them to adhere to the plastic for 2 hours. After this time i tried to remove them with long trypsin-EDTA treatment (10 min 37C) following by intensive up and down pipetation (i wanted to count the cells and place again a requested by my protocol amounts). However, they were adhered very strong so i lost about 50% of the cells.
somebody advice me to scrap the cells gently with a scraper. I wonder if the serum, gelatin or other covers could help to keep the cells safe from damage during the scrapping procedure? Is it this rationale after using HS???
all of the suggesting greatly will be greatly appreciated,
thank you in advance,
I think you have to trial and error on this. Decide what outcome measure is acceptable for your cells. I tried a cell scraper when I was working on pulmonary epithelial cells but was worried about the mechanical damage I might have been doing. There again, exposing the cells to a chemical environment harsh enough to shift them was a worry too. WIth each method I had to analyse the effect on the cells (mainly I used cell surface receptors and tested several drugs..ACh, noradrenaline, ATP etc). I used collagen coated plates and it worked a treat.
best of luck.