_{>> Cell Passaging <<}

Cell density estimations using a Neubauer hemacytometer is just particle counting. Additional information is required concerning the ration living particles at the total number. Consequently, a simple check for the ratio of vital cells is required. The staining of dispersed cells using the dye Trypan Blue before counting stains all dead cells blue (left image, a mix of living (white) and dead (blue) cardiac myocytes). Cells with an intact outer membrane do not accumulate the dye and stay white. This procedure is also applicable in transient cell culture to check for the living cells. Go here for the source of the image shown and also an application.

After seeding formerly fluent and round cells start to adhere (= stick) to the surfaces of the culture vessel. Also, they flatten and develop a cell specific shape. This adhesion process needs some hours. It can be suppressed by continuous agitation. This way in some cases it is possible to grow adherent cells in suspension. The figure righthand illustrates the adhesion process of freshly seeded fibroblasts.

Find in the following a short protocol about dissolving cells from a carrier with an digestion enzyme (trypsination), Trypan Blue staining for vitality check and new seeding. This complex is also called passaging. To be true: The time used to study protocoles is better spent in a cell culture lab together with an experienced instructor who demonstrates the procedure. Go also here.

Passaging:

Remove the culture medium and wash the dish or flask two times with Phosphate Buffered Saline (PBS) without calcium and Magnesium. This removes proteins which otherwise inactivate the digesting Trypsin solution. It is not senseful to give a particular trypsin concentration because this must compensate for special enzyme activity. Add trypsin solution and incubate for about five minutes either at room temperature or in the cell incubator. Check in the microscope: Trypsinized cells are round and volatile. Add a surplus of fresh medium to stop digestion. Estimate cell density using a hemacytometer and resuspend cell to a density between 5000 to 100.000 per ml.

Trypan Blue staining:

Add 1/10 aliquot Trypan Blue stock to the cell suspension, incubate for five minutes at room temperature and check in a hemacytometer. Caution: Trypan Blue is cytotoxic. So check immediately. After some hours any culture will yield a ratio of 100% dead cells.