Prepare a 1:1 dilution of the cell suspension in trypan blue: Approximately 10 microliters of cell suspension will be required to charge one chamber of the hemocytometer.For cells thawed from cryopreservation (in 1ml cryopreservation medium), pipette up and down 7-10 times using a one ml pipette. Pipette the cell suspension up and down in the tube 5-7 times using a pipette with a small bore (5 ml or 10 ml pipette). For an accurate cell count to be obtained, a uniform suspension containing single cells is necessary. Place the cell suspension in a suitably-sized conical centrifuge tube. Obtain a uniform suspension of cells: Follow the typsinization/trypsin neutralization protocol for the specific cell type.Inverted microscope (preferably phase contrast).0.4% Trypan Blue stain (fresh & filtered) in phosphate buffered saline.Hemocytometer plus a supply of cover slips.
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