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EDTA not working!!


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#1 themoon

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Posted 07 December 2009 - 02:51 PM

Hi,
I've tried various concentrations of EDTA (up to 0.04%) and my cells just won't come off. If I leave them in the incubator for about 10mins, they do come off, however I do notice a lot of cell damage. Any ideas? Should I increase the concentration up to 0.25%?

Many thanks

#2 bob1

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Posted 07 December 2009 - 02:55 PM

Just plain EDTA? Most people use trypsin(or other enzyme)-EDTA

#3 jah

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Posted 07 December 2009 - 06:55 PM

I agree w/ bob1 that most times a combination of Trypsin and EDTA is called for....unless you're staining for a surface marker. Assuming the latter case, have you washed your cells with Ca2+/Mg2+ free PBS or HBSS first? Usually a trypsin/EDTA solution has the EDTA at 0.53mM, (=155 g/l or 0.155%), so maybe more would help. It may not hurt to make sure the pH is near neutral, since EDTA solutions are generally rather alkaline (pH>8)

#4 themoon

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Posted 08 December 2009 - 06:05 PM

I agree w/ bob1 that most times a combination of Trypsin and EDTA is called for....unless you're staining for a surface marker. Assuming the latter case, have you washed your cells with Ca2+/Mg2+ free PBS or HBSS first? Usually a trypsin/EDTA solution has the EDTA at 0.53mM, (=155 g/l or 0.155%), so maybe more would help. It may not hurt to make sure the pH is near neutral, since EDTA solutions are generally rather alkaline (pH>8)



I was thinking of using the combo, however I think the tryp may be too harsh. I need to keep the surface proteins intact. Yes, I wash my cells with PBS free of divalent cations. Hmm I think I may need to try EDTA at 0.1% then. What concentration is the trypsin at in the trypsin/EDTA combination?

Thanks very much for your help guys! Really appreciate it!




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