PBMC Immunophenotyping by Flow Cytometry

Introduction

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Peripheral blood mononuclear cell (PBMC) analysis by flow cytometry is a useful application in wide range of research and clinical studies including HIV research, cancer immunotherapy and fundamental studies of cytokine-based immune responses. PBMCs are commonly extracted from peripheral blood, bone marrow and umbilical cord through density gradient centrifugation or magnetic bead separation. These approaches are designed to separate leukocytes from other cellular components such as red blood cells (RBCs).

A common application of flow cytometry is PBMC immunophenotyping. This technique measures the expression of cell surface molecules known as the 'cluster of differentiation' (CD) markers to identify, differentiate and characterize sub-populations of PBMCs. Beyond CD marker expression, precise monitoring of counts and cell sizes can be essential to this analysis. One such example is in the area of chimeric antigen receptor T (CAR-T) cell cancer immunotherapy.

In order to characterize CD markers on PBMCs, antibodies targeting CD markers are conjugated to a fluorophore such as phycoerythrin (PE). The fluorescence intensity is then measured using a flow cytometer to quantify CD marker expression levels and to differentiate PBMC sub-populations. This application note demonstrates both single and double fluorophore PBMC staining techniques used to characterize CD markers on PBMCs. CD45 and mouse IgG conjugates were prepared using PE and allophycocyanin (APC) Buccutite™ antibody labeling kits.

Human Peripheral Blood Mononuclear Cells (PMBC) Preparation

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Cryopreserved human PBMCs were thawed and prepared as follows:

Materials:

• Human PBMCs (Cryopreserved. 10 million cells/vial, iXCells Biotechnologies Cat No. 10HU-003)
• Blood Cell Culture Medium (iXCells Biotechnologies Cat No. MD-0007)
• Fetal Bovine Serum (Millipore Sigma Cat No. F4135)
• 15 mL conical tube
• Centrifuge
• Pipette

Method

1. Upon receipt of frozen PBMCs, thaw the cells and initiate the culture immediately in order to retain highest cell viability.
2. To thaw cells, place vial in 37 °C water bath with gentle stirring for ~1 minute.
   1. Note*: Keep cap out of the water to minimize risk of contamination.
3. Pipette the cells into a 15 mL conical tube with 10 mL fresh Blood Cell Culture Medium containing 10% Fetal Bovine Serum.
   1. Note*: Total Volume = 10.1 mL (100 µL PBMCs + 9 mL fresh Blood Cell Culture Medium + 1 mL Fetal Bovine Serum)
4. Centrifuge at 1,000 rpm (~220g) for 5 minutes at room temperature.
5. Remove the supernatant. Cells are ready to use.

Single and Double Fluorophore Staining of PBMCs:

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Conjugates and PBMCs prepared in the previous two sections were used in the following cell surface immunolabeling protocol.

Materials:

• PBMCs (iXCells Biotechnologies Cat No. 10HU-003)
• HHBS buffer (AAT Bioquest Cat No. 20011)
• Assay buffer (HHBS buffer with 1% BSA)
• Trypan Blue, sodium salt *UltraPure grade* (AAT Bioquest Cat No. 2452)
• TruStain FcX™ (BioLegend, Cat No. 422302)
• CD45 – PE Conjugates
• CD45 – APC Conjugates
• CD4 – FITC Conjugates (BioLegend Cat No. 317408)
• CD3 – APC Conjugates (BioLegend Cat No. 300312)
• Mouse IgG – PE Conjugates
• Mouse IgG – APC Conjugates
• NovoCyte 3000 Flow Cytometer (ACEA Biosciences, Inc.)

PBMCs Staining:

1. Prior to immunolabeling, determine PBMC viability using the Trypan Blue Dye Exclusion Test (AAT Bioquest Cat No. 2452) and 1 million cells were used per sample.
   1. For assistance use the Trypan Blue Dye Exclusion Test Application Note
2. Wash PBMCs with HHBS buffer (AAT Bioquest, Cat No. 20011) 2 times by centrifuge at 1000 RPM for 5 minutes each wash.
3. To block non-specific Fc-mediated interactions, re-suspend PBMCs in Assay Buffer (HHBS buffer containing 1% BSA solution) with Human TruStain FcX™ (BioLegend, Cat No. 422302) 5 µL/100 µL of Assay Buffer and incubate at room temperature for 10 minutes.
4. For single fluorophore PBMC staining, incubate PBMCs with CD45-PE, CD45-APC, Mouse IgG-PE or Mouse IgG-APC at a final concentration of 0.1 µg for 20 mins on ice, protected from light.
5. For double fluorophore PBMC staining, co-incubated PBMCs with CD45-PE alongside CD4-FITC and CD45-PE alongside CD3-APC. All four dyes had final concentrations of 0.5 µg for 20 mins on ice, protected from light.
6. Wash PBMCs with Assay Buffer twice and then resuspend PBMCs in Assay buffer.
7. Analyze on flow cytometer.

The NovoCyte 3000 flow cytometer (ACEA Biosciences, Inc.) was used for the analysis, and a total of 20,000 events were recorded. As shown in the Figure 1, based on the Forward Scatter and the Side Scatter, the lymphocytes were gated and staining intensity was measured for the gated populations using NovoCyte 3000 flow cytometer. For the PE and FITC combined staining, compensation was performed using single fluorophore stains and adjusted the overlap between those fluorophores.

In summary, Conjugates generated using Buccutite™ conjugation technology provides high quality yield. These conjugates were tested on PBMCs and are flexible with combining other conjugates for multicolor staining for detection of CD markers in PBMCs.

 

References

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1. Furtado, M. R. et al. Persistence of HIV-1 Transcription in Peripheral-Blood Mononuclear Cells in Patients Receiving Potent Antiretroviral Therapy. N. Engl. J. Med. 340, 1614–1622 (1999).
2. Kochenderfer, J. N. et al. Eradication of B-lineage cells and regression of lymphoma in a patient treated with autologous T cells genetically engineered to recognize CD19. Blood 116, 4099 LP-4102 (2010).
3. Haase, A. et al. Generation of Induced Pluripotent Stem Cells from Human Cord Blood. Cell Stem Cell 5, 434–441 (2009).
4. Bocelli-Tyndall, C. et al. Bone marrow mesenchymal stromal cells (BM-MSCs) from healthy donors and autoimmune disease patients reduce the proliferation of autologous- and allogeneic-stimulated lymphocytes in vitro. Rheumatol. 46, 403–408 (2007).
5. O'Sullivan, D. & Pearce, E. L. Targeting T cell metabolism for therapy. Trends Immunol. 36, 71–80 (2015)