Protocol A: Protocol of immunofluorescence staining of cells in combination with propidium

iodide staining of cells for cell cycle analysis

Reagents

 PBS
 70% ethanol
 2% (w/v) paraformaldehyde in PBS
 0.1% saponin (w/v)
 Propidium iodide (PI)
 Nucleic acid staining solution (1x PBS, 100 ug/mL RNAse A)
Methods

1. Prepare cells appropriately. Please refer to Cell Preparation for Flow Cytometry.
2. Fix in 2 mL 2% paraformaldehyde for 30 min on ice.
3. Centrifuge at 500 x g for 5 min. Discard supernatant.
4. Resuspend in 5 mL pre-cold 70% ethanol. Add dropwise to cell pellet while vortexing. Fix for
at least 30 min on ice.
(Specimens can be left at this stage for several weeks.)

5. Centrifuge at 500 x g for 5 min. Discard supernatant.

6. Wash twice with 0.1% saponin at 400 g at 4°C for 5 min. Discard the supernatant.
7. Resuspend in 100 mL of 0.1% saponin. Add the directly conjugated antibody at the vendor-
recommended dilution and incubate for at least 30 min at 4°C. Avoid direct light.
8. Wash twice in PBS. Pellet cells at 400 g at 4°C for 5 min. Discard supernatant.
9. Resuspend in 500 uL nucleic acid staining solution and incubate for 30 min at RT. Avoid
direct light.
10. Add appropriate PI (1-2 drops).
11. Analyze cells by flow cytometry.
12. Data analysis.
Notes

1. Protocol A only provides a general procedure for DNA staining for cell cycle analysis using PI
when you need to stain for other intracellular antigens.
2. Appropriate controls should always be carried out. For flow cytometry, the following should
be considered for inclusion.
o Isotype controls used to determine if the staining is specific.
o Unstained cells should always be included in the experimental set-up to monitor
autofluorescence.
3. For all multi-color flow cytometry experiments, it is suggested to include compensation
controls and Fluorescence Minus One (FMO) controls, which assist with identifying gating
boundaries.
Protocol B: Protocol of propidium iodide staining of cells for cell cycle analysis

Reagents
  PBS
 70% ethanol
 Nucleic acid staining solution (1x PBS, 100 ug/mL RNAse A)
 Propidium iodide (PI)
Methods

1. Prepare cells appropriately. Please refer to Cell Preparation for Flow Cytometry.
2. Fix in 5 mL pre-cold 70% ethanol. Add dropwise to cell pellet while vortexing. Fix for at least
30 min on ice.
(Specimens can be left at this stage for several weeks.)

3. Centrifuge at 500 x g for 10 min. Discard supernatant.

4. Wash twice with 3mL PBS at 400 g at 4°C for 5 min. Discard supernatant.
5. Resuspend cell pellet in 500 uL nucleic acid staining solution. Mix thoroughly.
6. Incubate for 30 min at RT.
7. Add appropriate PI (1-2 drops).
8. Analyze cells by flow cytometry.
9. Data analysis.
Notes

1. For Protocol B, unstained cells should always be included in the experimental set-up to
monitor autofluorescence.
Protocol C: Protocol of BrdU staining of cells for cell cycle analysis

Reagents

 PBS
 PBS-BSA: PBS containing 1% BSA
 0.05% (v/v) Tween 20 in PBS-BSA
 0.1M Na2B4O7, pH 8.5
 2M HCl containing 0.5% Triton X-100
 Propidium iodide (PI)
Methods

1. Add BrdU to the cell suspension in culture medium to a final concentration of 10 μM.
Incubate for at least 30 min at 37°C in a CO2 incubator.
2. Wash cells twice with PBS-BSA at 500 x g for 10 min at RT. Discard supernatant.
3. Resuspend in 5 mL pre-cold 70% ethanol. Add dropwise to cell pellet while vortexing. Fix for
at least 30 min on ice.
4. Centrifuge at 500 x g for 10 min. Discard supernatant.
5. Resuspend the pellet in 2 mL 2 M HCl containing 0.5% Triton X-100. Incubate for 30 min at
RT
(This incubation step is preferably performed on a rocking platform.)
 6. Centrifuge at 500 x g for 10 min. Discard supernatant. Resuspend in 3 mL 0.1 M Na 2B4O7, pH
8.5 for 2 min.
7. Centrifuge at 500 x g for 10 min. Discard supernatant. Resuspend in PBS-BSA containing
0.05% Tween 20.
8. Adjust cell concentration to 1x107 cells/mL. Aliquot 100 μL of the cell suspension into
required number of flow cytometry tubes.
9. Incubate with antibody at the recommended vendor dilution overnight at 4°C. Avoid direct
light.
10. Resuspend in 2 mL PBS-BSA. Centrifuge at 500 x g for 10 min.
11. If a secondary antibody is required, then discard the supernatant, add 100 μL of PBS-BSA
and incubate with the secondary antibody at the vendor recommended dilution for at least 30
min at 4°C.
12. Wash with 2 mL PBS-BSA. Centrifuge at 500 x g for 10 min.
13. Resuspend cells in 1 mL PBS.
14. Add appropriate PI (1-2 drops).
15. Analyze cells by flow cytometry.
16. Data analysis.
Notes

1. The acid treatment to unwind the DNA may affect surface immunophenotyping. Staining of
cells with BrdU using DNAse I may be applicable if this is required.
2. Appropriate controls should always be carried out. For flow cytometry, the following should
be considered for inclusion.
o A known positive sample
o Isotype controls used to determine if the staining is specific
o Unstained cells should always be included in the experimental set-up to monitor
autofluorescence
3. For all multi-color flow cytometry experiments, it is suggested to include compensation
controls and Fluorescence Minus One (FMO) controls, which assist with identifying gating
boundaries.

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 10 7 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 10 7 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)

o Remove the culture medium. Eliminate residual serum by rinsing cell monolayers
with PBS.
o Slowly add 1x Accutase solution or 0.25% Trypsin to cover the cell monolayer.
o Incubate at 37°C for up to 10 minutes.
o After incubation, gently tap the flask and the cells will detach and slide off in one
sheet to the bottom of the flask.
o 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.