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Flow Cytometry

Introduction

Flow Cytometry is a technology that measures and analyzes several physical characteristics of single cells. The cells flow in a fluid stream through a beam of laser light. Using Flow Cytometry the following cell characteristics can be determined: cell size, cell granularity, complexity and relative fluorescence intensity.

Flow Cytometry Intracellular and Membrane Staining Protocol

Cell fixation (for membrane protein):

  • Suspend cells in 1x PBS buffer and wash them twice with 1x PBS buffer by centrifugation at 1000 rpm for 5 min each time. Discard the supernatant.
  • Re-suspend the cells in 1 ml of 1x PBS buffer briefly.
  • Fix the cells in a final concentration of 4% formaldehyde (or paraformaldehyde)  for 20 min at room temperature.
  • Wash the cells 3 times with 1x PBS buffer by centrifugation at 1000 rpm for 5 min  each time.

Cell fixation and Permeabilization (for intracellular protein):

  • Permeabilize cells by adding 100% cold methanol slowly to pre-chilled  cells to a final concentration of 90% methanol before incubating for 30 min on ice.
  • Alternatively, fix the cells in a final concentration of 4% formaldehyde  (or paraformaldehyde) for 20 min at room temperature. Then incubate the cells  in 0.1% Triton X-100 in 1x PBS buffer for 15 min at room temperature.
  • Wash the cells 3 times with 1x PBS buffer by centrifugation at 1000 rpm for 5 min each time.

Immunostaining:

  • Blocking: Incubate the cells with 3 ml blocking buffer for 1h at room temperature.
  • Add primary antibody at an appropriate dilution and incubate for 1h at  room temperature.
  • Wash the cells 3 times with 1x PBS buffer by centrifugation at 1000 rpm for 5  min each time.
  • Add diluted secondary antibody (enzyme or fluorescein conjugated or other types)  to the cells and incubate for 1h at room temperature.
  • Wash the cells 3 times with 1x PBS buffer by centrifugation at 1000 rpm for 5  min each time.
  • Re-suspend the cells in 0.5 ml 1x PBS buffer and analyze the results on a flow cytometer. For DNA staining, re-suspend the cells in 0.5 ml of DNA dye instead;  incubate for at least 5 min at room temperature before analyzing the results on  a flow cytometer.

Buffers needed:

Blocking Buffer 1000 ml
Bovine serum albumin 5.00 g
1x PBS buffer 1000 ml

 

PBS Buffer 1000 ml
10 mM Na₂HPO₄ 1.42 g
1.7 mM NaH₂PO₄ 0.20 g
140 mM NaCl 8.19 g
Add ddH₂O to 1000 ml
Adjust to pH 7.4