I had problems in obtaining a good CD45 positive cell population after digestion with Liberase. I used same Liberase based on published protocol, the digestion time of 50 mins,37C, and similar concentration. I predigested it with 5 mM EDTA, 30 mins, 37C with constant shaking, rinsed 2x with PBS and thereafter subjected to Liberase + Dnase digestion. Then, depleted RBC using commercial RBC lysis buffer for 1 min. I tried it both for non-diseased (hemostasis) and mild disease (not diarrheic) but still most of the cells are either non CD45 live cells or dead cells. Any insights on this? Thank you.
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