Introduction

This protocol describes the immunophenotyping of cells in a whole blood sample by flow cytometry with minimal sample manipulation, thereby preserving cell structure and function while also reducing cell loss [1,2]. Whole blood samples stained with antibodies for cell-surface markers can be analyzed directly on a flow cytometer. To facilitate this analysis, red blood cells (RBCs) in the whole blood sample can be lysed after antibody staining. The ratio of RBCs to mononuclear cells in whole blood is approximately 600:1 (depending on sample and species); thus, when RBCs are present, the acquisition of many more events is required to observe rare cell events [1,3,4]. After RBC lysis, cells in the whole blood sample can be permeabilized and stained with antibodies for intracellular markers for subsequent analysis by flow cytometry.


Materials


Procedures

Whole blood preparation

  1. Collect whole blood into heparinized tubes or into tubes containing 1 μL 10% potassium EDTA per 100 μL of whole blood.
  1. For each antibody staining experiment, aliquot 100 μL unlysed whole blood into a 12 x 75 mm tube. For each single-color control (used to set compensation), aliquot 100 μL unlysed whole blood into a 12 x 75 mm tube. Reserve additional unlysed whole blood for an unstained control sample.

Cell surface staining

  1. Prepare desired antibody cocktail—containing fluorophore-labeled primary antibodies for cell-surface markers—in Flow Cytometry Staining Buffer. We recommend testing antibody dilutions from 1:50 to 1:100 initially. Protect from light.
  1. Add the antibody cocktail to a 100 μL aliquot of whole blood.
  1. Incubate for 30 minutes to 1 hour at 2–8°C with rotation. Protect from light.
  1. Wash samples with 2 mL Flow Cytometry Staining Buffer. This wash step can be repeated.
  1. Centrifuge at 500 x g for 5 minutes at 4°C. Discard the supernatant and resuspend in 500 μL Flow Cytometry Staining Buffer.

    Optional 5b: (Do not include this step when using fixation or lysis reagents.) An impermeant nucleic acid dye such as a SYTOX Dead Cell Stain can be added to gate viable cells. Do not add a wash step after adding this impermeant nucleic acid dye. Add 0.5 μL of SYTOX Dead Cell Stain solution per 500 μL sample, and mix well. Incubate for 20 minutes at room temperature, protected from light.
  1. Optional: Filter cells at room temperature through 0.45 μm cell strainer to remove large debris if running directly on a flow cytometer.


Optional: Red blood cell lysis

  1. For 100 μL of whole blood, add 2 mL of room temperature 1X 1-Step Fix/Lyse Solution, and invert gently. Lysing red blood cells creates easier gating conditions during flow cytometry analysis, though it can potentially interfere with immune cell function. The 1-Step Fix/Lyse Solution is formulated to lyse non-nucleated erythrocytes while maintaining a fixed and labeled leukocyte population.
  1. Incubate for 15 to 60 minutes at room temperature, protected from light.
  1. Store samples in buffer for up to 48 hours at 2–8°C, protected from light. Single color-stained samples, which are used to set compensation, and an unstained sample should be stored under the same conditions.

Protocol tip:

As compared with the antibody concentrations required in staining protocols for lysed blood samples or serum, the antibody concentrations used in whole blood staining protocols should be higher.

Protocol tip:

The No-Wash, No-Lyse Detection of Leukocytes In Human Whole Blood on the Attune NxT Flow Cytometer application note provides an example of gated, stained leukocytes.

Flow cytometry analysis

  1. Analyze samples on a flow cytometer equipped with appropriate filters, corresponding to the selected fluorophore labels.
  1. To achieve a CV of 1–10%, a minimum of 107–105 cell events must be acquired to find an event that occurs at a frequency of 0.1% in the cell population. The acquisition rate should be set to slowly collect events (e.g., 200–500 μL/min collection rate on the Invitrogen Attune Flow Cytometers).

Protocol tip:

The concentration of white blood cells varies from 4,000 to 10,000 cells per microliter (3). We recommend calculating the number of events required for a statistically significant result to estimate the amount of blood needed for an experiment (5).

Intracellular staining

  1. Before staining with antibodies that recognize intracellular markers, we recommend lysing the red blood cells and fixing the leukocyte population as described above using 2 mL 1X 1-Step Fix/Lyse Solution per 100 μL of whole blood. Invert gently, and incubate for 15 to 60 minutes at room temperature, protected from light.
  1. Dilute 10X Permeabilization Buffer to 1X by mixing 1-part 10X concentrate with 9 parts distilled water.
  1. Centrifuge sample at 500 x g for 5 minutes at room temperature. Discard the supernatant.
  1. Resuspend the pellet with 2 mL 1X Permeabilization Buffer, and centrifuge at 500 x g for 5 minutes at room temperature. Discard the supernatant. Repeat once. Protect from light.
  1. Resuspend the cell pellet with 100 µL of Flow Cytometry Staining Buffer.
  1. Prepare desired antibody cocktail—containing labeled primary antibodies for intracellular markers—in Flow Cytometry Staining Buffer. Protect from light. We recommend trying antibody dilutions from 1:50 to 1:100 initially.
  1. Add the antibody cocktail to the cell suspension.
  1. Incubate for 20–60 minutes at room temperature with rotation. Protect from light.
  1. Wash samples with 2 mL Flow Cytometry Staining Buffer. This wash step can be repeated.
  1. Centrifuge at 500 x g for 5 minutes at 4°C. Resuspend with 500 µL Flow Cytometry Staining Buffer.
  1. Analyze samples by flow cytometry.

Protocol tip:

Fixation and permeabilization may affect antibody specificity. Refer to the Intracellular Staining Buffer Selection Guide and specifically the "Fixation & Permeabilization" column for a non-exclusive list of antibodies that will work under fixation conditions.


References

  1. Petriz J, Bradford JA, Ward MD (2018) No lyse no wash flow cytometry for maximizing minimal sample preparation. Methods 134–135: 149–163. PMID 29269150.
  2. Rico LG, Juncà J, Ward MD et al. (2019) Flow cytometric significance of cellular alkaline phosphatase activity in acute myeloid leukemia. Oncotarget 10(65):6969–6980. PMID 31857851.
  3. Blumenreich MS. (1990) The White Blood Cell and Differential Count. In: Walker HK, Hall WD, Hurst JW (editors), Clinical Methods: The History, Physical, and Laboratory Examinations. 3rd edition. Boston: Butterworths; 1990. Chapter 153.
  4. Song S, Goodwin J, Zhang J et al. (2002) Effect of contaminating red blood cells on OKT3-mediated polyclonal activation of peripheral blood mononuclear cells. Clin Diagn Lab Immunol 9(3):708–712. PMID 11986282.
  5. Allan AL, Keeney M (2010) Circulating tumor cell analysis: Technical and statistical considerations for application to the clinic. J Oncol 2010:426218. PMID 20049168.