Roy J. Carver
Biotechnology Center

 Cell Preparation for Flow Cytometry

Depending upon your type of cells you need to transfer them from a medium and perform cell count and viability tests.  

1. Harvest the cells (if obtaining from tissue), decant (if grown in the flask) and centrifuge them for 4-5 minutes (300-400xg) at 4°C and discard the supernatant.
2. Resuspend the pellet in PBS or serum-free medium.
3. Centrifuge for 4-5 minutes (300-400xg)
4. Resuspend the pellet and perform cell count and viability analysis.

You can use hemocytometers for cell viability and cell count. You can use our automatic cell counter in the 231 ERML.  

1. Count the cells in squares 1,2,3,and 4.
2. Record, average and multiply by dilution factor (if cells were not diluted, then it is 10^⁴/mL).

Example:

1-60 cells; 2-66cells; 3-58 cells; 4-68 cells; Average – 63 cells

63 cells * 10^⁴/mL = your concentration

In order to determine cell viability you need to stain your cells either with trypan blue (positive test will illuminate blue) or erythrosin B (positive test will illuminate red). In flow cytometry, you can use fluorescent dye – Propidium Iodide to determine cell viability.

1. Mix your cell suspension 1:1 with erythrosin B solution in PBS or 0.4% trypan blue solution in PBS. 
2. Load the cells with erythrosin B solution to hemocytometer, but remember to incubate the cells with trypan blue for 2-5 minutes before loading
3. Nonviable cells will be stained red (erythrosin B) or dark blue (trypan blue). Cell viability is expressed as a percentage of unstained cells divided by the total number of cells.

We offer users cell sorting in two different ways:

1. Tubes (up to 4-way)
2. Well plates format.

All work performed by the Roy J. Carver Biotechnology Center (CBC) should be acknowledged in scholarly publications, posters, and presentations. Proper recognition allows us to measure the impact of our work and supports our initiatives in obtaining sponsored funding. In addition, any CBC personnel who make a substantial intellectual or experimental contribution are deserving of further recognition as co-author.