A small amount of trypan blue-cell suspension is transferred to one of the chambers of the hemocytometer by carefully touching the cover slip at its edge with the pipette tip and allowing each chamber to fill by capillary action. Ensure the cover slip and hemocytometer are clean and grease-free (use alcohol to clean). Load the hemocytometer: Moisten and affix cover slip to the hemocytometer.Pipette up and down several times to ensure a uniform cell suspension using the same pipette tip and allow to stand for 5-15 minutes. Combine the 10 microliters of cell suspension with the 10 microliters of trypan blue in the microfuge tube. Gently swirl (finger vortex) the cell suspension and remove 10 microliters of it using sterile technique. In a conical microfuge tube, add 10 microliters of 0.4% trypan blue solution.

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.

Burker Turk Counting Chamber Manual