I am interested to see the interferon secretion and RNA extraction from these harvested macrophages to see its further in-vitro action on HIV infected cells. Chemical method may interfere in signaling pathways, hence we decided to harvest by scrapping. We have tried by same method but count is very scanty.
Please help me to get maximum number of macrophages from THP- cells by your experience or std protocol.
Upon PMA treatment, THP-1 cells differentiate and attach tightly to cell culture plates. Therefore, detachment of cells from plates in good shape requires some experience and a good quality rubber policeman. Leave 1-2 ml medium (or PBS) in the plate and scrape off cells gently and slowly with policeman blade at ~45 degree angel. We routinely harvest cells without significant loss of cell viability.
Alternative method is to mature THP to macrophages in Teflon containers. Cells do have typical macrophage phenotype and don't adhere to Teflon. We published it in J. Immunol in 2009.
I agree with Divaker. When you want to harvest the cells after the treatment, you could use PBS (2ml) and put it on incubation at 37°C for 5 minutes with constant agitation. (or PBS + medium for 10-15min). This simple procedure makes the cells detach from the surface of the culture plates (or bottles). Then, sratch them softly, or take the medium and repeat.
Thanking you both of them to give me valuable suggestion and experience sharing. I will try accordingly and lets acknowledge as per.
I used to harvest primary human macrophages by incubation with Sigma non enzymatic cell dissociation solution (i think its based on a PBS EDTA solution) then very gently scraping the cells using a cell scraper (pull the scraper along the surface without putting any pressure on it)
If you plan to extract RNA from these cells why not seed and differentiate the cells in the plate/wells you plan to extract the samples from. This will minimise manipulation of the cells.
If THP-1 cells have not been matured for too long you can work with Accutase I. It works quite nice for mature THP-1 cells. We have used Accutase for sevaral years as part of our transfection protocol to detach premature THP-1 macrophages for nucleofection as well as for FACS analyses. In our hands, Accutase treatment is efficient, causes no or only a moderate increase in apoptosis and does not affect antibody-binding for FACS analyses.
we use Cellstripper (Cellgro), a non-enzymatic cell dissociation reagent that is a proprietary blend of chelators, to gently detach macrophages-including PMA-matured THP-1s- from tissue culture dishes. Incubate the cell monolayer with a small volume, as you would with trypsin, for up to 10 minutes before gentle scraping. We find this improves viability over scraping in PBS or media alone. Another good alternative for culturing macrophages -especially primary macrophages- is to plate them in regular plastic Petri dishes instead of tissue culture treated dishes. They do not attach as strongly and are much easier to detach by scraping without damage. We differentiate and culture the cells in Petrie dishes, then harvest, count, and transfer them to whatever size tissue culture plate is appropriate for our experimental protocol. Good luck!
I recommend using Cell Scraper from Corning - Costar catalog # 3010. These scapers are sterile 25 cm handle with 1.8 cm blade. I use it all the time and do not harm or activate my cells.
- Badges
- Science method