BestProtocols: Staining Cell Surface Targets for Flow Cytometry

Cell-surface markers can be used to define cell subsets based on lineage and developmental stage, as well as function when they are labeled with fluorochrome-conjugated antibodies and analyzed by flow cytometry. These surface markers have different forms and functions and include receptors for both soluble and cell-bound ligands, ion channels, glycoproteins, phospholipids, and more. For example, CD4 is a surface marker for T helper cells that can be further differentiated based on expression of other chemokine receptors and cluster of differentiation (CD) markers. Live cells stained with antibodies can be sorted based on unique staining patterns and used for additional experiments.

• Protocol A: Cell Suspensions
• Protocol B: Human Lysed Whole Blood

• For optimal performance of fluorochrome conjugated antibodies, store vials at 2–8°C in the dark. Do not freeze.
• Prior to use, quick spin the antibody vial to recover the maximum volume. Vortexing the antibody vial is not recommended.
• Except where noted in the protocol, all staining should be done on ice or at 2–8°C with minimal exposure to light.
• If storage of samples is necessary after staining with fluorophore-conjugated antibodies, store the samples in eBioscience IC Fixation Buffer (Cat. No. 00-8222-49) by combining 100 μL of sample with 100 μL of Fixation Buffer, or add 2 mL of eBioscience 1-step Fix/Lyse Solution (10X) (Cat. No. 00-5333-54). Samples can be stored in these buffers for up to 3 days in the dark at 2–8°C.
  • Note: There appears to be minimal impact on brightness of staining or FRET efficiency/compensation of tandem dyes, such as Invitrogen APC-eFluor 780 or PE-Cyanine7, when using the eBioscience IC Fixation Buffer or eBioscience 1-step Fix/Lyse Solution for storage of samples. Differences in fixation buffer quality can affect fluorophore brightness or FRET efficiency. Some generalizations regarding fluorophore performance after fixation can be made, but clone-specific performance should be determined empirically.
  • Note: Staining with fixable viability dyes is recommended before fixing samples to allow for gating on live cells during analysis by flow cytometry.

Materials

• Microcentrifuge tubes: 1.5mL (Fisher Cat. No. 05-408-136)
• 12 x 75 mm round-bottom test tubes (Fisher Cat. No. 22-171-606) or U- or V-bottom microplates (Fisher Cat. No. 08-772-54)
• Primary antibodies (directly conjugated or purified)
  • Secondary detection reagents, if primary antibodies are not directly conjugated
• eBioscience Flow Cytometry Staining Buffer (Cat. No. 00-4222-26)
• Required fluorophore buffers:
  • Super Bright Complete Staining Buffer (Cat. No. SB-4401-75) or Brilliant Stain Buffer (Cat. No. 00-4409-42)
• Optional: For blocking non-specific Fc-mediated interactions
  • Anti-mouse CD16/32 antibody (e.g., CD16/CD32 Monoclonal Antibody (93), (Cat. No. 14-0161-81)
  • Anti-human Fc receptor binding inhibitor antibody (e.g., Fc Receptor Binding Inhibitor Polyclonal Antibody, (Cat. No. 14-9161-71)
  • Normal Mouse Serum (Cat. No. 10410)
  • Normal Rat Serum (Cat. No. 10710C)
• Viability staining
  • Fixable stains: Invitrogen LIVE/DEAD Fixable Dead Cell Stain Kit (Cat. No. L34960)
  • Non-fixable stains:
    • eBioscience 7-AAD Viability Staining Solution (Cat. No. 00-6993-50)
    • eBioscience Propidium Iodide Staining Solution (Cat. No. 00-6990-50)
    • SYTOX Dead Cell Stains (Cat. No. S34862)

Experimental procedure

Note: Antibody-binding kinetics are temperature-dependent. Staining on ice may require longer incubation times. Furthermore, some antibodies may require non-standard incubation conditions that will be noted on the technical data sheet provided with the antibody.

1. Prepare cells as described in BestProtocols: Cell Preparation Protocols for Flow Cytometry.
2. Optional: Block non-specific Fc-mediated interactions. This is necessary when working with neutrophils, monocytes, macrophages, B-cells, natural killer cells, and some T cell subsets. Positive staining with anti-CD16/32 on cells will show cells that express Fc-gamma receptor.
   • For mouse cells: Pre-incubate the cells with 0.5–1 μg of anti-Mouse CD16/CD32 antibody per 100 µL for 10–20 minutes at 2–25°C before staining. Resuspend in 50 µL of eBioscience Flow Cytometry Staining Buffer.
   • For human cells: Pre-incubate the cells with 20 μL of anti-human Fc receptor binding inhibitor antibody per 100 µL for 10–20 minutes at 2–25°C before staining. Resuspend in 50µL of eBioscience Flow Cytometry Staining Buffer.
   • Pre-incubate the cells with 2 µL of 2% mouse serum to human cells and 2 µL of 2% rat serum to mouse cells per 100 µL of cells. Incubate for 15 minutes at 2–25°C. Do not wash prior to staining cells with antibodies.
     • Note: Mouse and rat serum are both required when using NovaFluor dyes with mouse cells.
3. Aliquot 50 µL of cell suspension (from 10⁵-10⁸) to each tube or microwell plate.
   • Tip: To determine the size of the sample (number of cells) that will provide a given precision, the equation r=(100/CV)x2 is used, where r is the number of cells and CV is the co-efficient of variation of a known positive control.
4. Block cells with the buffers associated with Super Bright dye, Brilliant Violet dye, Brilliant Ultra Violet dye, and/or Novafluor dye. Antibody mixtures should be made and used fresh.

When using antibodies labeled with polymer dyes (e.g., Super Bright dyes, Brilliant Violet dye, Brilliant Ultra Violet dye), block non-specific dye interactions by adding 5 µL Super Bright Complete Staining Buffer directly to the previously aliquoted cells in tubes or microplate wells; Super Bright Complete Staining Buffer can instead be added directly to tubes or microwell plates before the cell suspension is aliquoted. After adding buffer to cell suspensions, mix well by pipetting up and down or gently vortexing.

Note: Super Bright Compete Staining Buffer is only necessary when using more than one polymer dye-conjugated antibody in the same sample to minimize non-specific polymer dye interactions, which can result in data appearing undercompensated. Super Bright Complete Staining Buffer is compatible with traditional fluorophores.

Note: Brilliant Stain Buffer or Brilliant Stain Buffer Plus can be used as an alternative to Super Bright Complete Staining Buffer. It is not necessary to add both.

When using antibodies labeled with NovaFluor dyes or cyanine-based dyes, we recommend adding CellBlox Plus or CellBlox Blocking Buffer to antibody cocktails/master mixes prior to combining with cells. Add 5 µL per sample (regardless of the number of NovaFluor dyes in your panel) to use the antibody cocktail as intended. CellBlox Plus or CellBlox Blocking Buffer is required every time a NovaFluor dye is used with cells.

5. Combine the recommended quantity of each primary or fluorophore-labeled antibody in an appropriate volume of Flow Cytometry Staining Buffer so that the final staining volume is 100 µL (i.e., 50 µL of cell sample + 50 µL of antibody mix) and add to cells. Pulse vortex gently to mix.
6. Incubate for 30 minutes at 2–8°C. Protect from light.
   • Note: When using purified or biotinylated antibodies, incubate for one hour at 2–8°C.
7. Wash the cells by adding Flow Cytometry Staining Buffer. Use 2 mL per tube or 200 µL per microplate well. Centrifuge at 400–600 x g for 5 minutes at room temperature. Discard supernatant.
8. Repeat Step 7.
   • Note: Proceed to Step 12 if all primary antibodies were directly conjugated to fluorophores.
9. Dilute the appropriate fluorophore-labeled secondary detection reagent in 100 µL of Flow Cytometry Staining Buffer and add to cells. Incubate for at least 30 minutes at 2–8°C or on ice. Protect from light.
10. Wash the cells by adding Flow Cytometry Staining Buffer. Use 2 mL per tube or 200 µL per microplate well. Centrifuge at 400–600 x g for 5 minutes at room temperature. Discard supernatant.
11. Repeat Step 7.
12. Optional: Stain samples with a viability dye according to LIVE/DEAD staining protocol or BestProtocols: Viability Staining Protocol for Flow Cytometry.
    • Note: Use a LIVE/DEAD Fixable Dead Cell Stain with NovaFluor dyes. Impermeant DNA-binding dyes, such as DAPI, propidium iodide, and SYTOX stains are not compatible with NovaFluor dyes.
13. Optional: For storage of samples before analysis, resuspend cells in 100 µL of Flow Cytometry Staining Buffer and add 100 µL of IC Fixation Buffer or 2 mL of 1-step Fix/Lyse Solution.
    • Note: SYTOX reagents, 7AAD, and propidium iodide are not compatible with cells that have been fixed. Resuspend cells in an appropriate volume of Flow Cytometry Staining Buffer (i.e., 10⁵ cells in microtiter plate will be resuspended in less than 500 µL of buffer.)
14. Analyze samples by flow cytometry analyzer or isolate cells with a flow cytometry cell sorter.

Either the eBioscience 10X RBC Lysis Buffer (multi-species) or eBioscience 1-step Fix/Lyse Solution (10X) may be used to lyse red blood cells (RBC) after whole blood has been stained with fluorophore-conjugated antibodies. Additionally, the 1-step Fix/Lyse Solution lyses RBC and fixes leukocytes in a single step. If lysed whole blood cells will be prepared using the 1-step Fix/Lyse Solution before staining, confirm that the antibodies in the staining panel recognize fixed epitopes on the antigens of interest. Please refer to the Antibody Fixation Considerations: Antibody clone performance following fixation for information on antibody clone performance before and after fixation.

Materials

• Microcentrifuge tubes: 1.5 mL (Fisher Cat. No. 05-408-136)
• 12 x 75 mm round-bottom test tubes (Fisher Cat. No. 22-171-606) or U- or V-bottom microplates (Fisher Cat. No. 08-772-54)
• Anticoagulant, such as:
  • Heparin (Fisher Cat. No. BP252410)
  • (K2 or K3) Potassium EDTA
• eBioscience 10X RBC Lysis Buffer (Multi-species) (Cat. No. 00-4300-54) or eBioscience 1-step Fix/Lyse Solution (10X) (Cat. No. 00-5333-54)
  • Note: Before using, the 10X RBC Lysis Buffer (Multi-species) and 1-step Fix/Lyse Solution (10X) must be diluted to 1X by adding 1 part buffer with 9 parts room temperature, reagent-grade water.
• Primary antibodies (conjugated or unlabeled and purified)
  • Secondary detection reagents, if primary antibodies are not directly conjugated
• eBioscience Flow Cytometry Staining Buffer (Cat. No. 00-4222-26)
• Required fluorophore buffers:
  • Super Bright Complete Staining Buffer (Cat. No. SB-4401-75) or Brilliant Stain Buffer (Cat. No. 00-4409-42)
  • Invitrogen CellBlox Plus Blocking Buffer (Cat. No. C001T06F01) or CellBlox Blocking Buffer (Cat. No. B001T06F01); CellBlox Plus is recommended for improved NovaFluor dye staining performance.
• Optional: Block non-specific Fc-mediated interactions
  • Anti-mouse CD16/32 antibody (e.g., CD16/32 Monoclonal Antibody (93) (Cat. No. 14-0161-81)
• Anti-human Fc receptor binding inhibitor antibody (e.g., Fc Receptor Binding Inhibitor Polyclonal Antibody (Cat. No. 14-9161-71)
• Normal Mouse Serum (Cat. No. 10410)
• Normal Rat Serum (Cat. No. 10710C)
• Viability Staining
  • Fixable stains: Invitrogen LIVE/DEAD Fixable Dead Cell Stain Kit (Cat. No. L34960)
  • Non-fixable stains:
    • eBioscience 7-ADD Viability Staining Solution (Cat. No. 00-6993-50)
    • eBioscience Propidium Iodide Staining Solution (Cat. No. 00-6990-50)
    • SYTOX Dead Cell Stains (Cat. No. S34862)

Experimental procedure

Note: Antibody-binding kinetics are temperature-dependent. Staining on ice may require longer incubation times. Furthermore, some antibodies may require non-standard incubation conditions that will be noted on the technical data sheet provided with the antibody.

1. Collect whole blood into heparinized tubes or into tubes containing 1 µL 10% potassium EDTA per 100 µL of whole blood.
2. Aliquot 100 µL of whole blood to each tube or microplate well.
3. Optional: Block non-specific Fc-mediated interactions. This is necessary when working with neutrophils, monocytes, macrophages, B-cells, natural killer cells, and some T cell subsets. Positive staining with anti-CD16/32 on cells will show cells that express Fc-gamma receptor. Pre-incubate the whole blood with 20 μL of anti-human Fc receptor binding inhibitor antibody per 100 µL of blood. Incubate for 10–20 minutes at 2–25°C before staining.
4. Block cells with the buffers that are associated with Super Bright dyes, Brilliant Violet dyes, Brilliant Ultra Violet dye, and/or Novafluor dyes. Antibody cocktails should be made and used fresh.

When using antibodies labeled with polymer dyes (e.g., Super Bright dyes, Brilliant Violet dye, Brilliant Ultra Violet dye), block non-specific dye interactions by adding 5 μL Super Bright Complete Staining Buffer directly to the previously aliquoted cells in tubes; Super Bright Complete Staining Buffer can instead be added directly to tubes before the cell suspension is aliquoted. After adding buffer to cell suspensions, mix well by pipetting up and down or gently vortexing.

Note: Super Bright Compete Staining Buffer is only necessary when using more than one polymer dye-conjugated antibody in the same sample to minimize non-specific polymer dye interactions, which can result in data appearing undercompensated. Super Bright Complete Staining Buffer is compatible with traditional fluorophores.

Note: Brilliant Stain Buffer can be used as an alternative to Super Bright Complete Staining Buffer. It is not necessary to add both.

When using antibodies labeled with NovaFluor dyes or cyanine-based dyes, add 5 µL of CellBlox Plus or CellBlox Blocking Buffer to the previously aliquoted cells in tubes or microplate wells. CellBlox Plus or CellBlox Blocking Buffer can instead be added directly to tubes or microplate wells before the cell suspension is aliquoted. After adding buffer to cell suspensions, mix well by pipetting up and down or gently vortexing. This required every time a NovaFluor dye is used with cells.

5. Combine the recommended quantity of each fluorophore-labeled antibody in an appropriate volume of Flow Cytometry Staining Buffer so that the final volume is 50 µL and add to whole blood. Pulse vortex gently to mix.
6. Incubate for 20–30 minutes at 2–25°C. Protect from light.
   • Note: Proceed to Step 10 if all primary antibodies were directly conjugated to fluorophores.
7. When using purified or biotinylated antibodies, incubate for one hour at room temperature.
8. Wash the cells by adding Flow Cytometry Staining Buffer. Use 2 mL per tube or 200 µL per microplate well. Centrifuge at 400–600 x g for 5 minutes at room temperature. Discard supernatant.
9. Dilute the appropriate fluorophore-labeled secondary detection reagent in 100 μL of Flow Cytometry Staining Buffer and add to the cells. Incubate for 15–30 minutes at 2–8°C or on ice. Protect from light.
10. Without washing cells, add 2 mL of freshly prepared 1X RBC Lysis Buffer and pulse vortex briefly. Incubate for 10–20 minutes at room temperature. Protect from light.
    • Note: Do not incubate longer than 20 minutes if using the 1X RBC Lysis Buffer.
11. Centrifuge at 400–600 x g for 5 minutes at room temperature. Discard supernatant.
12. Wash the cells by adding Flow Cytometry Staining Buffer. Use 2 mL per tube or 200 µL per microplate well. Centrifuge at 400–600 x g for 5 minutes at room temperature. Discard supernatant.
13. Repeat Step 12.
14. Optional: Fixable viability dyes may be used to stain whole blood before using the 1-step Fix/Lyse Solution. Stains samples with a viability dye according to LIVE/DEAD staining protocol or BestProtocols: Viability Staining Protocol for Flow Cytometry.
    • Note: Use a LIVE/DEAD Fixable Dead Cell Stain with NovaFluor dyes. Impermeant DNA-binding dyes such as DAPI, propidium iodide, and SYTOX stains are not compatible with NovaFluor dyes.
    • Note: Viability dyes including SYTOX reagents, propidium iodide, or 7-AAD should not be used on cells lysed using the 1-step Fix/Lyse Solution. Fixation can permeabilize cells.
15. Resuspend cells in an appropriate volume of Flow Cytometry Staining Buffer (i.e., 10⁵ cells in a microtiter plate will be resuspended in less than 500 µL of buffer).
16. Analyze samples by flow cytometry or isolate cells with a flow cytometry cell sorter.