Flow Cytometry Sample Preparation

Preparation of cells for staining

Note: This information is to serve as a guide as individual investigators may need to optimize protocols for their particular cell type.

Cells for flow cytometry analysis are usually derived from four main sources:

• Adherent or suspension cultures
• Cryopreserved samples
• Whole blood or buffy coats
• Solid tissues e.g. bone marrow, spleen, intestine etc.

Regardless of the source, the final cell preparation should be:

• a homogenous single-cell suspension free of clumps and dead cell debris
• at a density of 10⁶-10⁷ cell per ml
• suspended in a suitable staining buffer

PBMCs isolated from whole blood through Ficoll gradient centrifugation or RBC lysed whole blood or non-adherent cultured cells are readily available for flow cytometric analysis. Adherent cultured cells or cells present in the solid organs should be first made into a single cell suspension before flow analysis by using enzymatic digestion or mechanical dissociation of the tissue, respectively. Mechanical filtration should be followed to avoid unwanted instrument clogs or lower quality flow data. Use the following sample preparation protocols based on your appropriate starting materials:

1. Preparation of suspension culture cells
2. Preparation of adherent culture cells
3. Preparation of cryopreserved cells
4. Preparation of cells from blood or buffy coat
5. Preparation of cells from murine bone marrow

Preparation of suspension culture cells

Key Reagents – PBS, staining buffer

1. Decant cells from tissue culture vessel into centrifuge tube(s).
2. Centrifuge at 300-400 x g for 5-10 min at room temperature.
3. Discard supernatant and re-suspend pellet in PBS and repeat previous step.
4. Discard supernatant and re-suspend in suitable volume of cold staining buffer.

Preparation of adherent culture cells

Key Reagents – PBS, staining buffer, 0.25% trypsin

1. Discard culture medium and rinse the cell monolayer with sterile PBS
2. Add warmed trypsin to just cover the monolayer and incubate at 37°C for 5-10 min (depending on the cell type).
3. Neutralize the reaction with culture medium (serum added) and detach the cells by gently shaking the vessel.
4. Continue as with suspension culture cells preparation protocol.

Preparation of cryopreserved cells

Key reagents – PBS, staining buffer, culture medium with 10% FBS

1. Thaw the cryo-tubes rapidly in a water bath set at 37°C.
2. Transfer to a chilled centrifuge tube and add ice cold culture medium drop by drop until the cells are diluted 10X. Perform on ice!
3. Centrifuge at 300-400 x g for 5 min at 4°C.
4. Discard supernatant and wash once with cold staining buffer.
5. Re-suspend cells in a suitable volume of cold staining buffer.

Preparation of cells from blood or buffy coat

Key reagents – PBS, staining buffer, suitable gradient medium like Ficoll or Histopaque

1. Dilute whole blood or buffy coat with equal volume of room temperature PBS.
2. Carefully layer the diluted blood over an equal volume of the gradient medium.
3. Centrifuge at 400-500 x g for 30-40 min at room temperature. The centrifuge brakes should be turned off!
4. Aspirate the PBMCs from the thin interface between the upper plasma layer and lower medium layer.
5. To remove granulocytes, aspirate the whitish colored layer just above the RBC sediment [Note: specialized gradient media have been formulated to enrich for different granulocyte populations].
6. Re-suspend the cells in PBS and centrifuge at 300-400 x g for 10 min. at room temperature.
7. Wash with PBS once or twice more.
8. Re-suspend the cells in a suitable volume of staining buffer.

Preparation of cells from murine bone marrow

Key reagents – PBS, staining buffer (see appendix), RBC lysis buffer [check Appendix for recipe or use a commercially available buffer]

1. Dissect out the tibia and femurs and remove all extraneous fat, muscle and connective tissue. Put the bone in cold PBS and store on ice – perform all following steps on ice.
2. Fill up a syringe with cold culture medium and fit an 18 gauge needle to it. Drill the ends of the bones with the needle and flush out the contents onto a non-tissue culture treated plate.
3. Break up the cell masses into a single cell suspension with the help of a 22-gauge needle.
4. Pellet down the cells at 300-400 x g and wash once more with cold PBS.
5. Re-suspend the cells in a suitable volume of staining buffer and perform a cell count before pelleting the cells again.
6. Re-suspend the cells in RBC lysis buffer at 10⁶ cells/ml and incubate at room temperature for 5 minutes.
7. Pellet down the cells, discard the lysis buffer and wash once with PBS.
8. Re-suspend the cells in a suitable volume of staining buffer.