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Flow Cytometry Protocol: Sample Preparation

Single-cell suspension is required for flow cytometry assays. Thus the adherent cell lines and tissue samples require processing into single-cell suspension before flow cytometry analyzed. A number of protocols are available and involved in mechanical dissociation or enzymatic digestion of the sample.

Note: When an enzymatic digestion is carried out, incubation has to be carefully monitored.

Reagents:

  • PBS: Dissolve 8 g NaCl, 0.2 g KCl, 1.15 g Na2HPO4 and 0.2 g KH2PO4 in 800 mL distilled water. Adjust the pH to 7.4 with HCl and final volume to 1 liter with additional distilled H2O.
  • Cell staining buffer: Add 0.5% BSA and 0.05% Sodium Azide (NaN3) to PBS, sterile-filtered.
  • RBC lysis buffer: 150 mM NH4Cl, 10 mM KHCO3 and 500 μM EDTA, sterile-filtered.
  • Ficoll - Paque or other density separation medium
  • Enzymes for tissue digestion
  • Cell culture medium (serum added)
  • EDTA

Equipment:

  • 15-mL centrifugal tube
  • Tissue culture dish
  • Scissors and scalpel blade
  • Springe
  • Centrifuge

Procedures:

Protocol A: Cultured Cells

1. Cell cultured in suspension just need to be harvested and centrifuged at 300-500 x g for 4-5 minutes and washed three times with cell staining buffer to remove residual growth factors that may be present in the culture medium. Then skip to Step 7.

2. For adherent cell lines, they may require pretreatment with EDTA or trypsin to facilitate removal from their substrates. Cells treated by trypsin should be further incubated in cell culture medium (serum added) on a rocker platform to neutralize the enzymatic activity without reattachment to the substrates.

3. Filtrate cell suspension through a 40 μm cell strainer to eliminate clumps and debris.

4. Centrifuge at 400-500 x g for 3-5 minutes and discard the supernatant.

5. Wash cells with cell staining buffer. Centrifuge at 300-400 x g for 5 minutes at 4°C and discard supernatant.

6. Repeat Step 5 twice

7. Resuspend the pellet in cell staining buffer.

8. Take 100 μL for cell counting and viability analysis.

9. Adjust the suspension to a concentration of 1 x 106 cells/mL in cell staining buffer.

Protocol B: Tissue Sample

1. Harvest tissue into a tissue culture dish containing 10 mL PBS.

2. Cells are dissociated by mechanical trituration and enzyme digestion.

  • Mechanical trituration: Tissue is cut into 2-4 mm pieces by scissors and scalpel blade and triturated with the plunger of a springe.
  • Enzyme digestion: After mechanical trituration, cells are detached using 0.25% trypsin diluted in PBS for 20 minutes at room temperature.

3. Neutralize the reaction with cell culture medium (serum added).

4. Filtrate cell suspension through a 40 μm cell strainer to eliminate clumps and debris.

5. Centrifuge at 300-500 x g for 4-5 minutes at 2-8 °C. Discard the supernatant.

6. (Optional) If necessary, resuspend cells in 3 mL 1 X RBC lysis buffer and incubate on ice for 5 minutes to lyse erythrocytes. Stop the reaction with 10 mL cell staining buffer. Centrifuge at 300-400 x g for 5 minutes at 4°C and discard the supernatant.

7. Wash cells with cell staining buffer. Centrifuge at 300-400 x g for 5 minutes at 4°C and discard supernatant.

8. Repeat Step 7 twice.

9. Resuspend the pellet in cell staining buffer and perform a cell count and viability analysis.

10. Adjust the suspension to a concentration of 1 x 106 cells/mL in cell staining buffer.

Protocol C: Isolate PBMC from Whole Blood

1. Anticoagulated-blood is collected and diluted with PBS (1:1). Additionally, an equal volume of Ficoll underlay the diluted sample.

2. Centrifuge at 350 g for 10-20 minutes at room temperature.

3. Aspirate PMBC at the interface of the PBS and Ficoll layers.

4. Resuspend the cells in PBS.

5. Centrifuge at 300-500 g for 4-5 minutes at 2-8 °C. Discard the supernatant.

6. Resuspend cells in 3 mL1 X RBC lysis buffer and incubate on ice for 5 minutes to lyse erythrocytes. Stop cell lysis with 10 mL cell staining buffer. Centrifuge at 300-400 x g for 5 minutes at 4°C and discard the supernatant.

7. Wash cells with cell staining buffer. Centrifuge at 300-400 x g for 5 minutes at 4°C and discard supernatant.

8. Repeat Step 7 twice.

9. Resuspend the pellet with staining buffer and perform a cell count and viability analysis.

10. Adjust the suspension to a concentration of 1 x 106 cells/mL in cell staining buffer.

Learn the principle of flow cytometry.

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