Dear flower, I am working on Differentiation of k562 cell line. I want to set it,So I treated the cells (100,000/ml) with erythropoietin (it isn't for culture and it is for therapy ) in 5 and 10 U/ml with the final volume 5ml(4ml cells and 1ml epo). after 5 days I take slides from my cells with cytospin3 and staind with wright and DAB,but there wasn't any differentiation in my cells. DAB preparation:(diaminobenzidine) 1)I dissolved 20mg DAB in 25ml tris_buffer 1Molar. 2)I added to it 60mic H2O2 30%. 3)I put the slides that fixed in methanol for 10 min in DAB for 20 min. 4)after washing with D.W I staind them with counter stain (gimsa for 15 min) But there wasn't any differentiated cells. I would be glad you can help me. Your sincerely. Mahmoodzadeh. Tarbiat Modares University. Department of Hematology .
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