We usually use 1 mL 1X Trypsin/EDTA, colleagues from different departments use 2 mL and i have seen it work with 700 uL... But what is the ideal amount of Trypsin to use?
Based on the information in your question, it appears that you are referring to the volume of the Trypsin/EDTA solution that is used to split cell cultures in a plate/flask. If the information is correct, one needs to consider the following things: number of washes with PBS to remove previous medium that contains proteins (serum), temperature of the Trypsin/EDTA solution (4 or 37 degree C), and volume needed. After one wash of cells with PBS, Trypsin/EDTA solution at 4 degree C, and sufficient volume to cover cells by tilting the cell culture plate at least once or twice may be sufficient. Watch cells under the microscope for rounding and detach cells by gently stroking the plate. Quinch the Trypsin action by adding fresh medium supplemented with serum.
I agree with Dr. Choubey's excellent advice.
SH-SY5Y cells are not so difficult to handle. Make sure you:
-- have enough EDTA in the trypsin mix
-- introduce a brief (< 1 min) rinse step with trypsin (1-2 mL) over the cell surface (then discard it) immediately after PBS wash, to remove residual endogenous trypsin-inhibitors.
-- do the normal trypsinization afterwards.
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For typical 10-cm dish, 1-2 mL should be okay (but it depends also on your trypsin concentration -- whether it's 0.25 or 0.05%). Sometimes 1mL trypsin can be used, and the total volume be made up to 2mL with warm PBS. However, this way, the effective EDTA conc. falls.
Wash the cells well to remove trypsin-inhibiting serum (2X), add just enough trypsin (0.05%)/EDTA to cover the cells (1-2 ml or a 10 cm dish), and incubate at 37oC during digestion. The SH-SY5Y cells take a bit longer to lift than other common cell types, but are not particularly hard to handle. In our experience, temperature is a key issue in speeding the process and improving efficiency of the digestion. Good luck.
We use 1ml Trypsin(kept at water bath 37oC) for a 75 cm2 flask, after cells washed with 10 ml PBS . We incubate 1 minute at 37oC.When we remove flask from incubator, we agitate the flask with hand to detach cells.
Many thanks to all for the information. I will keep using 1 mL, which is just enough to cover the cells, and which easily detaches all the cells.
Since the cells do detach quite quickly, i thought maybe even less would be sufficient, thinking that too much trypsin may damage the surface proteins. But if this is the standard protocol, I will assume that the cells are not harmed.
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