So I am about to use Raw264.7 cell line in flow cytometry to measure the signal of an intracellular protein. The cells are know not to effectively detach with trypsin so I will use scraping. Will I have problems running the sample in the Flow Cytometer (Calibur) because of the presence of cell clusters? Can pipetting separate cells from each other? Is there any other method you have used?
Clumps usually create problem in FACS, I would pipette them well with low volume media and pass them through a sieve to remove the clusters and treat the single cells. Adding EDTA might help as well.
You could try this to avoid clumps
Corning Falcon Test Tube with Cell Strainer Snap Cap.
The following method could help you.
1. Aspirate all growth media the wash with 5 to 10 ml of PBS, gently shaking the PBS around the flask and aspirate
2. Eject half of culture volume of trypsin directly onto cells (the cells will need to be lifted at this point) and incubate for 5 minutes at 370C.
(RAW cells are quite adherent to tissue culture flask; if the trypsin does not immediately lift the cells, do not proceed to wait longer as this risks the viability of cells).
3. Eject (half of culture volume+0.5mL) warm media directly onto cells
4. Gently use the cell lifter and scrape cells off the flask. These cells do not lyse easily with a cell lifter. Collect the cell suspension from flask.
Is there some treatment with ice to harvest macrophages RAW 264.7?
Thanks
@Pau Torres Matrí I've read that 5mM EDTA for 10min in the fridge is a way of harvesting. It didn't work for me. I did the scrapping but afterwards I pipette like 15 times against the plate so I avoid clumps a lot and I get a fairly clear population at the flow cytometry
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