Hi, everyone, I want to isolate CD45 +ve and -ve cells from mouse stomatch. I tried once. Stomach was cutted into peices as small as I can, and then digested in RPMI1640 medium containing 20mM HEPES, 1mg/ml dispase, 1mg/ml collagenase A and DNase I, at 37c for 50min. After that, I moved the content into a 50ml tube through 100um cell strainer, with the remaining peices being meshed. However, many cells (>90%) were dead as shown during sorting. Hence, only got very few cells.
Do you know the optimal protocol?
Thanks a lot.
I don't know of an optimal protocol, but I will say that I've been happy so far with the Biomasher disposable mashing tubes for retrieving similar cells from less fibrous organs with a simple EDTA or heparin pre-treatment in PBS. Also, look into the PluriSelect cell strainers - you can put them on a 5 mL or even a 1.5 mL microcentrifuge tube. A colleague found these and we really like them so far, especially if we are working with low cell counts and don't want to dilute to a volume suitable for 50 mL conicals.
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