Flow Cytometry Protocols

Introduction Single-cell suspensions are required for all flow cytometry assays. Thus, peripheral blood cells or cells that grow in suspension are well suited for analysis by flow cytometry. Adherent cell lines, solid tissue samples, and tumors require processing into single-cell suspensions before they can be analyzed. Numerous protocols are available and may involve enzymatic digestion or mechanical dissociation of the tissue. Care should be used when chelation or enzymatic digestion are used, as these may result in the destruction of the antibody epitope. In all situations, removing cell clumps, dead cells, and debris is essential to eliminate false positives and obtain results of the highest quality. Materials • Gibco™ PBS (Phosphate-buffered saline), pH 7.4, (Cat. No. 10010023) • Ficoll-Paque™ PLUS medium (GE Healthcare, Cat. No. 17-1440-02) or other density separation medium • Flow Cytometry Staining Buffer (Cat. No. 00-4222) or other buffer of choice • 15- or 50-mL conical centrifuge tubes Experimental procedure 1. Dilute blood sample at least 1:1 with PBS in a conical tube. 2. Underlay the diluted sample with a volume of Ficoll that is equal to the original sample volume. 3. Centrifuge at 400 x g for 20 minutes at room temperature with the brake OFF. 4. Harvest PBMC located at the interface of the PBS and Ficoll layers into a fresh tube. 5. Fill the tube with PBS to wash the cells. 6. Centrifuge the cells at 300–400 x g for 4–5 minutes at 2–8°C. Discard supernatant. 7. Resuspend the cell pellet in an appropriate volume of Flow Cytometry Staining Buffer or buffer of choice and perform a cell count and viability analysis. 8. Centrifuge cells as in Step 4 and resuspend in appropriate volume of Flow Cytometry Staining Buffer or buffer of choice so that the final cell concentration is 1 x 107 cells/mL (other cell concentrations may be appropriate for different experiments). Isolation of human peripheral blood mononuclear cells from whole blood If cells are to be cultured, perform all steps using aseptic technique and buffers that do not contain azide. This protocol is intended for use with the specific products mentioned within it. Substituting different products is not recommended. Note Note 1For Research Use Only. Not for use in diagnostic procedures. Mouse bone marrow cell isolation Introduction This protocol describes the isolation of cells from the marrow of bone [1,2]. As compared with whole blood, bone marrow yields a more complex set of cells, including hematopoietic stem cells–responsible for the production of leukocytes, erythrocytes, and thrombocytes–and stromal cells, such as endothelial cells, fibroblasts, macrophages, osteoblasts, osteoclasts, and adipocytes. Materials • Cell culture dishes (e.g., Thermo Scientific Nunc EasYDish Dishes, 100 mm, Cat. No. 150466) • Lab wipes (e.g., Fisher Scientific Kimberly-Clark Professional Kimtech Science Kimwipes Delicate Task Wipers, 1-PlyFisher) • Ethanol (e.g., Absolute Ethanol, 200 Proof, Molecular Biology Grade) • RPMI Complete Media –RPMI 1640 medium (e.g., Gibco BenchStable RPMI 1640, Cat. No. A4192301) –2 mM L-glutamine (e.g., Gibco L-Glutamine, 200 mM, Cat. No. 25030149) –Fetal bovine serum (e.g., Gibco Fetal Bovine Serum, Cat. No. 26140087) –Optional Penicillin-streptomycin (e.g., Gibco Penicillin-Streptomycin (5,000 U/mL), Cat. No. 15070063, or Gibco Penicillin-Streptomycin (10,000 U/mL), Cat. No. 15140122) • Scalpel and blades • 5–10 mL syringe (e.g., Fisherbrand Sterile Syringes for Single Use, 10 mL) • 25-gauge needle (e.g., Fisher Scientific BD General Use and PrecisionGlide Hypodermic Needles) • 70 μm cell strainers (e.g., Fisherbrand Sterile Cell Strainers) • 50 mL conical tubes (e.g., Thermo Scientific Nunc 50 mL Conical Sterile Polypropylene Centrifuge Tubes, Cat. No. 339652) • Red blood cell (RBC) lysis buffer (e.g., Invitrogen eBioscience 1X RBC Lysis Buffer, Cat No. 00-4333-57 or Invitrogen eBioscience 10X RBC Lysis Buffer (Multi-species), Cat. No. 00-4300-54) • Cell counter (e.g., Invitrogen Countess 3 Automated Cell Counter, Cat. No. AMQAX2000) • Gibco™ PBS (Phosphate-buffered saline), pH 7.4, (Cat. No. 10010023) This protocol is continued on page 3 2Sample preparation For Research Use Only. Not for use in diagnostic procedures. Procedure Cell isolation 1. Under sterile conditions, isolate bone marrow femurs from mice, and place into a sterile cell culture dish. 2. Use forceps and small scissors to cut away the muscle and fibrous tissues from the bone. If muscles are still intact, wipe the bones with a Kimwipe saturated with 70% ethanol to remove any excess muscle fibers. 3. Add 5–7 mL RPMI complete media to a new sterile cell culture dish. Prepare complete RPMI 1640 medium by supplementing RPMI 1640 medium with fetal bovine serum to a final concentration of 10%, 2 mM L-glutamine (if using medium not currently supplemented with GlutaMAX). 4. Cut both femur ends with a scalpel or sharp scissors so that both ends are clean. 5. Attach a 25-gauge needle to a 10 mL syringe. Draw up 5 mL RPMI complete media into the syringe. 6. Using forceps, hold the bone above the cell culture dish containing 5–7 mL RPMI complete media, and carefully flush out the marrow into the cell culture dish using the syringe containing 5 mL RPMI complete. Repeat flushing as necessary until the bone is white, which indicates that all the marrow has been removed. Repeat this step for all bones. 7. Place a 70 µm cell strainer on top of a new 50 mL conical tube, held upright in a tube rack. 8. Using a sterile serological pipet, transfer all marrow from cell culture dish into the strainer. Add more RPMI complete media to the cell culture dish and continue transferring any leftover marrow to the strainer. 9. Using just the plunger from a 10 mL syringe (DO NOT TOUCH the black rubber end of the plunger; keep it sterile), mash the marrow contained in the 70 µm cell strainer in a downward circular motion. Verify there are no large pieces of bone marrow left. This protocol is continued on page 4 Supplement media with 1% penicillinstreptomycin (5,000 units/mL). Optional After flushing the femur bones, they may be crushed by mortar and pestle for additional cells. Bone marrow can also be isolated from crushed pelvic bones and tibias. Protocol tip 3For Research Use Only. Not for use in diagnostic procedures. Mouse bone marrow cell isolation, cont. 10. Rinse any leftover marrow from the plunger end with RPMI complete media into the strainer to collect as many cells as possible. 11. Rinse strainer with 5–7 mL RPMI complete media. Remove the strainer and cap the conical tube containing the sample. 12. Spin down cells at 600 x g for 4 minutes at 4°C. Discard the supernatant. 13. Add about 5 mL RPMI complete media to the cell pellet, and pipet up and down to resuspend. 14. Place a new 70 µm cell strainer on top of a new 50 mL conical tube in a rack. 15. Strain the resuspended marrow sample through the cell strainer and into the conical tube. Count viable cells if desired. 16. Resuspend cell sample to desired cell concentration with appropriate media. Cell counting 1. Lyse a small sample of the red blood cells with an RBC lysis buffer to easily count immune cells. Add 1 mL 1X RBC lysis buffer to 100 μL sample. 2. Incubate at room temperature for 4–5 minutes with occasional shaking or rotating. 3. Add 2–3 mL 1X PBS to stop the reaction. 4. Spin down the cells at 600 x g for 4 minutes at 4°C. Discard the supernatant. 5. Add about 500 μL complete RPMI to pellet, and pipet up and down to break the pellet. 6. Count viable cells with a hemocytometer or the Invitrogen Countess 3 Automated Cell Counter. Proceed to immune cell isolation protocols using Invitrogen MagniSort Kits or Invitrogen Dynabead Kits or to cell stimulation protocols. Optional 1. J Biol Methods (2014) 1(1):e1. 2. Bio Protoc (2015) 5(20):e1631. PMID 27441207. References 4Sample preparation For Research Use Only. Not for use in diagnostic procedures. Mouse spleen cell isolation protocol Introduction The spleen is the site for hematopoiesis, red blood cell clearance, and immunologic functions, and therefore, a good source of cells. It filters cell debris, pathogens, and irregular cells. It is a source for both red blood cells and leukocytes and for several immune cell subtypes including granulocytes, monocytes, macrophages, dendritic cells (DCs), NK cells, T cells, and B cells. Leukocytes can be found in the crude spleen preparation. DCs and macrophages can be isolated by enzymatic release of the cells from the crude cell preparation. Materials • Red blood cell (RBC) lysis buffer (e.g., eBioscience 10X RBC Lysis Buffer (Multispecies), Cat. No. 00-4300-54) • Gibco™ PBS (Phosphate-buffered saline), pH 7.4, (Cat. No. 10010023) • Scalpel and blades • Cell culture dishes (e.g., Thermo Scientific Nunc EasYDish Dishes, 100 mm, Cat. No. 150466) • Disposable transfer pipette (e.g., FisherBrand Disposable Graduated Transfer Pipettes) • 50 mL conical tubes (e.g., Thermo Scientific Nunc 50 mL Conical Sterile Polypropylene Centrifuge Tubes, Cat. No. 339652) • 70 μm cell strainers (e.g., Fisherbrand Sterile Cell Strainers) • 5–10 mL syringe (e.g., Fisherbrand Sterile Syringes for Single Use, 10 mL) • Hank’s balanced salt solution (e.g., Gibco Hank’s Balanced Salt Solution, HBSS 10X, Cat. No. 14060040) Preparation of myeloid cells • Collagenase (e.g., Gibco Collagenase, Type IV, Cat. No. 17104019) • DNase (e.g., Thermo Scientific DNase I solution, Cat. No. 90083) • Fetal Bovine Serum (e.g., Gibco Fetal Bovine Serum, Cat. No. 26140087, or Fetal Bovine Serum One Shot format, Cat No. A3160401) • EDTA (e.g., UltraPure 0.5M EDTA, pH 8.0, Cat. No. 15575020) This protocol is continued on page 6 5For Research Use Only. Not for use in diagnostic procedures. Procedure 1. Obtain fresh whole mouse spleen. 2. Place mouse spleen into petri dish with 5 mL HBSS buffer. 3. Carefully mince the spleen into small pieces (~0.2 cm2) with a razor or scalpel blade. 4. For preparation of myeloid cells (continue to step 5 for crude preparation): Incubate the excised spleen pieces for 20–30 min at 37°C with 5 mL of HBSS containing Collagenase IV (100 U/mL) and DNase (20 µg/mL) solution with 1% FBS. 5. Add EDTA 1 mM/mL for 5 minutes at room temperature to stop the enzymatic reaction. 6. Place cell strainer over a 50 mL conical tube. 7. With a disposable transfer pipette, transfer the excised spleen into the cell strainer. 8. With the plunger end of a syringe, mash or press the spleen through the strainer. Add 5–10 mL PBS if necessary. 9. Wash the cells through the strainer with excess PBS. Repeat step 6 and 7, if needed. 10. Centrifuge the cells at 400–600 x g for 5 minutes at 4°C; discard the supernatant. 11. Resuspend the cell pellet in 2–5 mL of cold 1x RBC Lysis buffer. 12. Incubate the suspension for 5 minutes on ice. 13. Wash the cell suspension with 10–20 mL cold PBS. 14. Centrifuge the cells at 400–600 x g for 5 minutes at 4°C; discard the supernatant. 15. Resuspend the cell pellet in PBS at 2–3 x 106 cells/mL. Perform steps 1–8 at room temperature and steps 9–13 on ice with cold buffers. Critical note Mouse spleen cell isolation protocol, cont. 6Sample preparation For Research Use Only. Not for use in diagnostic procedures. Whole blood staining protocol for flow cytometry analysis 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 • Blood collection tubes with anticoagulant, either heparin, K3EDTA, or K2EDTA tubes • 12 x 75 mm round-bottom polystyrene tubes (e.g., Thermo Scientific Samco 12 x 75 mm Disposable Culture Tubes, Cat. No. 12-007S) • Flow cytometry staining buffer (e.g., Invitrogen eBioscience Flow Cytometry Staining Buffer, Cat. No. 00-4222-26) • Fluorophore-labeled primary antibodies (e.g., validated Invitrogen Flow Cytometry Antibodies) • Gibco™ PBS (Phosphate-buffered saline), pH 7.4, (Cat. No. 10010023) • Red blood cell (RBC) lysis buffer (e.g., Invitrogen eBioscience 1X RBC Lysis Buffer, Cat No. 00-4333-57 or Invitrogen eBioscience 10X RBC Lysis Buffer (Multi-species), Cat. No. 00-4300-54) • Permeabilization buffer (e.g., Invitrogen eBioscience Permeabilization Buffer (10X), Cat. No. 00-8333-56) • Optional 0.45 µM cell strainer (e.g., Falcon Cell Strainer, Cat. No. 08-771-1) • Optional Non-fixable cell viability dye (impermeant nucleic acid dye) (e.g., Invitrogen SYTOX Dead Cell Stain Sampler Kit, for flow cytometry, Cat. No. S34862) • Optional Fix/Lyse solution (e.g., Invitrogen eBioscience 1-Step Fix/Lyse Solution (10X), Cat. No. 00-5333-54) This protocol is continued on page 8 7For Research Use Only. Not for use in diagnostic procedures. 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. 2. 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. 2. Add the antibody cocktail to a 100 μL aliquot of whole blood. 3. Incubate for 30 minutes to 1-hour at 2–8°C with rotation. Protect from light. 4. Wash samples with 2 mL Flow Cytometry Staining Buffer. This wash step can be repeated. 5. Centrifuge at 500 x g for 5 minutes at 4°C. Discard the supernatant and resuspend in 500 μL Flow Cytometry Staining Buffer. Optional (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. 6. Optional Filter cells at room temperature through 0.45 μm cell strainer to remove large debris if running directly on a flow cytometer. 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. 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. To access the application note, visit thermofisher.com/ nowashnolysewholeblood This protocol is continued on page 9 Whole blood staining protocol for flow cytometry analysis, cont. 8 Protocol tip Protocol tipSample preparation For Research Use Only. Not for use in diagnostic procedures. 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. 2. Incubate for 15 to 60 minutes at room temperature, protected from light. 3. 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. Flow cytometry analysis 1. Analyze samples on a flow cytometer equipped with appropriate filters, corresponding to the selected fluorophore labels. 2. 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). 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]. This protocol is continued on page 10 9 Protocol tipFor Research Use Only. Not for use in diagnostic procedures. Fixation and permeabilization may affect antibody specificity. Refer to the Intracellular Staining Buffer Selection Guide at thermofisher.com/ intracellularstainingbuffer and specifically the “Fixation & Permeabilization” column for a non-exclusive list of antibodies that will work under fixation conditions. 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. 2. Dilute 10X Permeabilization Buffer to 1X by mixing 1-part 10X concentrate with nine parts distilled water. 3. Centrifuge sample at 500 x g for 5 minutes at room temperature. Discard the supernatant. 4. 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. 5. Resuspend the cell pellet with 100 µL of Flow Cytometry Staining Buffer. 6. 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. 7. Add the antibody cocktail to the cell suspension. 8. Incubate for 20–60 minutes at room temperature with rotation. Protect from light. 9. Wash samples with 2 mL Flow Cytometry Staining Buffer. This wash step can be repeated. 10. Centrifuge at 500 x g for 5 minutes at 4°C. Resuspend with 500 µL Flow Cytometry Staining Buffer. 11. Analyze samples by flow cytometry. Whole blood staining protocol for flow cytometry analysis, cont. 1.Methods 134–135:149 (2018). PMID 29269150. 2. Oncotarget 10(65):6969 (2019). PMID 31857851. 3. Clinical Methods: The History, Physical, and Laboratory Examinations (1990) 3rd edition. Boston: Butterworths; 1990. Chapter 153. 4. Clin Diagn Lab Immunol 9(3):708 (2002). PMID 11986282. 5. J Oncol 2010:426218 (2010). PMID 20049168. References 10 Protocol tipSample preparation For Research Use Only. Not for use in diagnostic procedures. Red blood cell lysis Introduction Before using peripheral blood or certain lymphoid tissue suspensions for flow cytometric analysis or for in vitro assays, red blood cells (RBC) should be removed. The 1X RBC Lysis Buffer (Cat. No. 00-4333) and 10X RBC Lysis Buffer (Multi-species) (Cat. No. 00-4300) are formulated for optimal lysis of erythrocytes in single-cell suspensions of human peripheral blood and mouse tissue (such as spleen). The buffers contain ammonium chloride, which lyses RBC with minimal effect on leukocytes. When using human peripheral blood for flow cytometric analysis, the RBC lysing step can be incorporated into the staining protocol. The 1-step Fix/Lyse Solution (10X) (Cat. No. 00-5333) is formulated for the combined lysis of RBC and fixation of peripheral blood leukocytes after staining with fluorochrome-conjugated antibodies. All of the RBC lysis reagents are compatible with fluorochrome-conjugated antibodies. Red Blood Cell (RBC) lysis protocols • Protocol A: Using 1X or 10X RBC Lysis Buffers –A1 Antibody Staining Followed by Lysis of Whole Peripheral Blood –A2 Bulk Lysis of Human Whole Blood –A3 Lysis of Mouse/Rat Spleen or Bone Marrow Cells