I have tried using Mitlenyi's Gentlemacs with type III collagenase at 37C for about 45 minutes-60 minutes and I am fairly sure I am over-digesting the T cells. After processing to single cell suspensions, cells have poor viability and Thy1.2 isolation does not really enrich the T cells. Additionally, surface markers like CD62L and CD3 are being shed it appears.
Hello
See these paper and links may help you
Good Luck
http://www.jove.com/video/50088/live-imaging-drug-responses-tumor-microenvironment-mouse-models
http://www.bio-protocol.org/e1097
Hi Colt Egelston,
you have to follow the Mitlenyi's biotech protocol you will get enough of enrich the T cells and viability also good. try one more time very carefully.
HI,
even with the optimised Miltenyi's protocols i had to further optimise them for my own "needs" as my tissues (primary human breast tumours) were also overdigested even with their mildest protocol.
I would recommend to reduce the incubation times slightly which could prove to be beneficial for your T cell survival.
regarding the markers...being a spontaneous tumour may needs some characterisation.
good luck
Thanks everyone. We currently think that Liberase treatment for 30 minutes is the gentlest and most effective option.
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