Flow Cytometry


Flow cytometry is a technology for cell counting, cell sorting, specific biomarker measurement et cetera which base on laser application. The samples are cells and particles as usual, and they are suspended in a stream of fluid. Flow cytometry permits analysis of the physical and chemical characteristics at the same time for thousands of particles per second. Flow cytometry is widely used in basic research, diseases diagnosis and clinical practice. A common application is to physically sort cells based on specific properties so that we can purify cells which we are interested in.

Flow Cytometry protocol

Materials
Cells
PBS
Primary antibody
BSA
Centrifuge
Vortex
FACS Tubes

Cell culture

  1. Seed cells in culture plate or culture flask at an appropriate initial concentration.
  2. Culture cells in right medium at the recommend conditions for a period of time.
  3. Treat cells following your experimental design.

Procedure

  1. Wash the cells with ice-cold PBS twice by centrifugation to forbidden unknown interference if necessary.
  2. Harvest the cells by trypsin or other methods which are appropriate for you.
  3. Adjust cell concentration to 1-5 x 106 cells/mL in ice cold PBS. Cells will be stained in any container for which is suitable for your centrifuge e.g. test tubes, eppendorf tubes.
  4. Add 0.1-10 μg/mL of the labeled primary antibody in 3% BSA/PBS.
  5. Incubate for at least 30 min at room temperature or 4°C. The incubation time and temperature can be adjusted according to your requests.
  6. Wash the cells three times by centrifugation at 400 g for 5 min, discard the supernate.
  7. Resuspend cells in 500 µL ice cold PBS.
  8. Protect the cells from light and keep on ice or at 4°C in a fridge. We recommend you to analyze the cells as soon as possible.
  9. Transfer the cells to FACS Tubes and make sure homogeneity before the flow cytometry analysis.


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