Protocol of Cell Preparation for Flow Cytometry

Provided here are protocols for harvesting cells from various sources to obtain healthy cells, essential for optimal staining and analysis. Refer to the corresponding sections according to your experimental requirements.

Contents:

• Protocol A: Preparation of Human Peripheral Blood Mononuclear Cells
• Protocol B: Preparation of Tissue Culture Cells for Flow Cytometry
• Protocol C: Preparation of Peritoneal Macrophages, Bone Marrow, Thymus and Spleen Cells

Protocol A: Preparation of Human Peripheral Blood Mononuclear Cells

Reagents

• PBS
• PBS-BSA: PBS containing 1% BSA

Method

1. Allow separation media to equilibrate to RT.
2. Dilute blood in equal volumes of PBS-BSA.
(For instance, add 3 mL of PBS-BSA to 3 mL blood)
3. Carefully overlay whole blood onto an equal volume of separation media in a 15 mL conical centrifuge tube.
4. Centrifuge at 400g for 30 minutes in a 20°C temperature-controlled centrifuge with no brake.
(Centrifugation at 4°C or with brake reduces efficiency of cell recovery.)
5. Harvest cells from the serum/separation media interface using a pipet.
6. Place harvested cells in a 15 mL conical centrifuge tube.
7. Adjust the volume to 10 mL with PBS-BSA.
8. Centrifuge at 400 g for 5 minutes at RT.
9. Discard supernatant and resuspend pellet to a final concentration of at least 1 x 10⁷ cells/mL with pre-cold (4°C) PBS-BSA.

Protocol B: Preparation of Tissue Culture Cells for Flow Cytometry

Reagents

• PBS
• PBS-BSA: PBS containing 1% BSA
• 1x Accutase solution
• 0.25% Trypsin

Methods

Preparation of Cells Stored in Liquid Nitrogen

1. Carefully remove cells from liquid nitrogen storage.
2. Thaw cells rapidly in a 37°C water bath.
3. Resuspend cells in cold (4°C) PBS-BSA and transfer them to a 15 mL conical centrifuge tube.
4. Centrifuge at 400 g for 5 min at 4°C.
5. Discard supernatant and resuspend pellet in an appropriate amount of cold (4°C) PBS-BSA, such as 1x10⁷ cells/mL.
(Higher viability can be obtained by allowing the cells to recover in culture media overnight.)

Preparation of Tissue Culture Cell Lines in Suspension

1. Decant cells from tissue culture flask into 15 mL conical centrifuge tube.
2. Centrifuge at 400 g for 5 minutes at RT.
3. Discard supernatant and resuspend pellet in 10 mL of PBS-BSA.
4. Centrifuge at 400 g for 5 minutes at RT.
5. Discard supernatant and resuspend to a minimum concentration of 1 x 10⁷ cells/mL in cold (4°C) PBS-BSA.

Preparation of Adherent Tissue Culture Cell Lines

1. Harvest cells by enzymatic release using 1x Accutase solution or 0.25% trypsin, followed by quenching with media containing serum.
(Epitopes may be cleaved when using the enzymatic digestion method. Cells can also be harvested by gently scraping them into culture media)
• Remove the culture medium. Eliminate residual serum by rinsing cell monolayers with PBS.
• Slowly add 1x Accutase solution or 0.25% Trypsin to cover the cell monolayer.
• Incubate at 37°C for up to 10 minutes.
• After incubation, gently tap the flask and the cells will detach and slide off in one sheet to the bottom of the flask.
• Add growth medium and re-suspend the cells by gently pipetting.
2. Centrifuge at 400 g for 5 min.
3. Discard supernatant and resuspend pellet in fresh PBS-BSA to wash off any remaining cell debris and proteins.
4. Centrifuge at 400 g for 5 minutes at RT.
5. Discard supernatant and resuspend pellet in an appropriate amount of PBS-BSA.
6. Count cells using a hemocytometer or an automated cell counter. Dilute the cells with cold (4°C) PBS-BSA to a minimum concentration of 1 x 10⁷ cells/mL.

Protocol C: Preparation of Peritoneal Macrophages, Bone Marrow, Thymus and Spleen Cells

Reagents

• PBS
• PBS-BSA: PBS containing 1% BSA
• Ammonium chloride lysis buffer: 0.16M ammonium chloride, 0.17M Tris, pH7.2
• PBS-BSA with 25 μg/mL DNAse I or 5 mM EDTA

Methods

1. Prepare a single cell suspension from relevant tissue. Keep cells on ice to minimize cell death.
(Addition of DNAse I or EDTA can also reduce aggregation. Large aggregates can be removed by passing through a 40 μm cell strainer.)
2. Centrifuge at 400 g for 5 minutes at 4°C.
3. Discard supernatant and resuspend pellet in 10 mL ammonium chloride lysis buffer.
4. Mix and incubate for 2 minutes at 4°C. Do not exceed this time.
5. Centrifuge at 400 g for 5 minutes at 4°C.
6. Discard supernatant and resuspend pellet in 10mL cold (4°C) PBS-BSA.
7. Centrifuge at 400 g for 5 minutes at 4°C.
8. Discard supernatant and resuspend pellet to a final volume of 10mL with cold (4°C.) PBS-BSA.
9. Count cells using a hemocytometer or an automated cell counter. Dilute the cells with cold (4°C) PBS-BSA to a minimum concentration of 1 x 10⁷ cells/mL.

Notes

1. In order to avoid non-specific binding, it is necessary to block FcR on cell types such as spleen cells with FcR blocking reagents.
2. These methods offer a general procedure for use with peripheral blood mononuclear cells, single cells derived from tissue culture cell lines, cell suspension, cells acquired from the peritoneum / bone marrow / thymus / spleen. These are guidelines only and may need to be adjusted for particular applications.