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Applications

protocols

We've prepared this section as a guide to help you prepare various cell types for analysis using flow cytometry. Use it as a starting point and expect that it will require further optimization for your investigations.

Cell Preparation of Tissue Culture Cells

Materials

Enzyme Cell Detachment Medium, Trypsin or EDTA (10 mM in PBS)
Sony Flow Cytometry Staining Buffer(Cat. No. 2701005)
15 or 50 mL conical centrifuge tubes

Procedure

Note: If you are using cells that grow in suspension, you need to decant the cells in a conical centrifuge tube then perform a cell count and viability analysis. In this case you should proceed to Step 3.

  1. For adherent cells lines, there are pros and cons of different materials to detach the cells from the plate. EDTA is preferred as will have minimal effect on protein staining (except where the epitope is modified by the removal of calcium ions). Scraping can result in cell clumps which are undesirable for flow cytometry and Trypsin needs to be tested empirically before proceeding to ensure it will not destroy the epitope of the protein you are staining. In general, 0.025 to 0.5% trypsin is the dynamic range and each cell line may have different requirements, enzyme Cell Detachment Medium can also be used, but can alter some cell surface epitopes; the effect will need to be determined empirically for the epitopes being evaluated.
  2. Place cells into a conical centrifuge tube and perform a cell count and viability analysis.
  3. Centrifuge cells and re-suspend in an appropriate volume of Flow Cytometry Staining Buffer so that the final cell concentration is 1x107 cells/mL.

Cell Staining

Preparation of samples for Cell Surface Staining

Suspension cells

Materials

  • Sony Cell Staining Buffer (Cat. No. 2701005)
  • 15 or 50 mL conical centrifuge tubes
  1. Transfer to a conical tube and count number of viable cells.
  2. Centrifuge cells at 350 x g for 5 min.
  3. Discard supernatant and resuspend in an appropriate volume of Cell Staining Buffer so that the final cell concentration of cells is 5 – 10 x106 cells/mL.

Adherent cells

Materials

  • Detachment media such as PBS containing pH balanced PBS with trypsin EDTA
  • Sony Cell Staining Buffer (Cat. No. 2701005)
  • 15 or 50 mL conical centrifuge tubes
  1. Detach cells using determined detachment buffer. Gentle scraping may also be used. However, it may not sufficiently separate the cells.
  2. Transfer to a conical tube and count number of viable cells.
  3. Centrifuge cells at 350 x g for 5 min.
  4. Discard supernatant and resuspend in an appropriate volume of Cell Staining Buffer so that the final cell concentration of cells is 5 – 10 x106 cells/mL.

Isolations of PBMCs (peripheral blood mononuclear cell) from whole blood

For best results prepare sample within two hours of blood draw

Materials

  • Ficoll® Paque or other density separation medium
  • Phosphate buffered saline (PBS) or other balance salt solution (not containing magnesium or calcium)
  • Sony Cell Staining Buffer (Cat. No. 2701005)
  • 15 or 50 mL conical centrifuge tubes
  1. Place blood sample in a conical tube and dilute at least 1:1 with PBS.
  2. In a separate tube place and amount of Ficoll® equal to the original blood sample volume.
  3. Gently pipet the diluted blood sample on top of the Ficoll® taking care not to mix the layers.
  4. Centrifuge the sample for 20 – 30 min at 400 x g at room temperature with no brake.
  5. Carefully remove the PBMCs located at the interface of the PBS and Ficoll® layers and transfer into a fresh tube. Take care to collect as little Ficoll® and diluted plasma as possible.
  6. Remove contaminating Ficoll® by washing cells. Fill tube to the top with PBS.
  7. Centrifuge cell suspension for 5 -10 minutes, 300 x g at a temperature of 4°C, discard supernatant.
  8. Resuspend the cell pellet in Cell Staining Buffer and count the number of viable cells.
  9. Centrifuge cells as in Step 7, then resuspend cells in an appropriate volume of Cell Staining Buffer to a final concentration of ~1x107 cells/mL.

Preparation of Cells from Lymphoid Tissue (spleen, lymph nodes, thymus)

Mechanical disruption combined with cells straining is typical sufficient to obtain a single cell suspension

Materials

  • 60 mm x 15 mm tissue culture dish
  • 3 mL syringe
  • Cell strainer (nylon mesh) (available from many suppliers)
  • Sony Cell Staining Buffer (Cat. No. 2701005)
  • 15 or 50 mL conical centrifuge tubes
  1. Dissect out tissue and place into a tissue culture dish and break it apart into a single cell suspension by pressing with the plunger of a 3 ml syringe.
  2. Add 10 mL of Cell Staining Buffer and pass resulting cell suspension through a cell strainer to remove clumps and debris.
  3. Place cell suspension into a fresh conical tube (note many cell strainers are designed to sit on top of a conical tube).
  4. Centrifuge cell suspension at 300 X g for 5 -10 minutes at 4°C, remove and discard supernatant.
  5. Add 10 ml of staining buffer and gently resuspend the cells.
  6. Count the number of viable cells.
  7. Centrifuge cells as in Step 4, then resuspend cells in an appropriate volume of Cell Staining Buffer to a final concentration of ~1x107 cells/mL.

Non-Lymphoid Tissue Preparation

The ability to prepare a single cell suspension varies greatly by tissue type. Combinations of mechanical disruption and enzymes are commonly used. For best results for your specific tissue type we recommend consulting the literature.

Antibody Cell Surface Staining Protocol

For optimal performance primary antibodies should be titrated individually. A good starting concentration is that recommended by the manufacturer. When using a panel of multiple antibodies it is best to prepare a cocktail rather than dispensing individual labeled antibodies into tubes. FMO controls (fluorescence minus one - cocktail minus each one of the labeled antibodies) are strongly recommend when developing panels.Under certain conditions use of an Fc blocking solution can reduce nonspecific background. For example staining mouse samples with mouse antibodies or human samples with antibodies of mouse IgG2a isotype.
Materials

  • Sony Cell Staining Buffer (Cat. No. 2701005)
  • 12 X 75 mm round bottom tubes or other vessel
  • Human TruStain FcX™ (Fc Blocking Solution) (Cat. No. 2711505) or TruStain FcX™ (anti-mouse CD16/32) (Cat. No. 1106595), if required.
  • Fluorochrome labeled primary antibodies
  1. Store all samples and reagents on ice to prevent internalization of receptors.
  2. Add 100 μl of cell suspension to each tube.
  3. If using blocking solution use 5 μl of Human TruStain FcX™ or 1 μg of TruStain FcX™ per 106 mouse cells. It is not necessary to remove the blocking agent prior to primary incubation with primary antibody.
  4. Add the appropriate amount of labeled primary antibody. If the primary antibody needs to be diluted use Cell Staining Buffer as the diluent.
  5. Incubate for 15 – 60 minutes on ice in the dark. Incubation times should also be optimized.
  6. Fill tube fill Cell Staining buffer, centrifuge at 250 x g for five min., discard supernatant, and repeat.
  7. Resuspend cells in 0.2 to 0.5 ml Cell Staining Buffer. Store samples at 4°C in the dark. Analyze cells as quickly as possible after staining.*

*If cells need to be preserved for several days add 0.5 ml of Fixation Buffer (Cat. No. 2704005) after step 5. Let incubate at room temperature for 20 min in the dark. Continue with step 6.

Intracellular Secreted Proteins

Intracellular and secreted proteins

Why study intracellular and secreted proteins by flow cytometry?

Unlike Western Blots or ELISAs flow cytometry allows researchers to determine:

  • Which subpopulations of cells are expressing the protein of interest
  • Relative distribution of secreted protein within a population. For example are all cells secreting the same amount or are a few cells secreting large quantities?
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Special considerations must be made when detecting a secreted or intracellular proteins by flow cytometry. These include challenges with the antibody accessing the antigen and keeping the protein to be secreted inside the cell. All protocols to stain intracellular proteins require some level of cell permeabilization. Cell permeabilization is typically achieved by detergents and alcohol, which can alter or destroy cell surface antigens. Therefore the most gentle method should be used to maintain the integrity of surface molecules. In some cases researchers may choose to stain surface markers prior to fixing and permeabilizing cells.

Detection of cytokines and other secreted proteins by flow cytometry

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Secreted proteins by definition are outside the cell. To trap the protein inside the cell protein transport inhibitors, which interfere with the function of the Golgi apparatus, are used. Cells are then fixed and gently permeabilized before staining with fluorchrome conjugated antibodies.It is important to note that prior stimulation with agents such as PMA/ionomycin is usually required for detection of cytokines.1 The optimal choice of stimulant and protein transport inhibitor will vary by sample type and application. Please consult the literature for best options.

Materials

  • Activating agent such as PMA (50 ng/ml)/Ionomycin (1 μg/ml) or LPS (100 ng/ml)
  • Brefeldin A Solution (1,000X) (Cat. No. 2703005) or Monensin Solution (1,000X) (Cat. No. 2703505)
  • Cell Staining Buffer (Sony Cat. No. 2701005)
  • Antibodies to cell surface markers conjugated to fluorochromes (if desired)
  • Fixation Buffer (Sony Cat. No. 2704005)
  • Antibodies to intracellular proteins directly conjugated to fluorochromes
  • Intracellular Staining Permeabilization Wash Buffer (10X) (Sony Cat. No. 2705010). It is especially important to include negative controls such as isotype controls as intracellular staining often has higher backgrounds.
  • Deionized water (DI)
  • 15 or 50 mL conical centrifuge tubes
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Stimulate cells for approximately 4-6 hours depending on application. Add Monensin or Brefeldin A to block protein transport.
  1. Harvest and prepare cells according to appropriate protocol for surface staining.
  2. Centrifuge cells at 350 x g for 5 min.
  3. Discard supernatant and resuspend in an appropriate volume of Cell Staining Buffer so that the final cell concentration of cells is 5 – 10 x106 cells/mL.
  4. Add the appropriate amount of labeled primary antibody to cell surface markers. If the primary antibody needs to be diluted use Cell Staining Buffer. Alternatively all cell surface markers can be stained along with intracellular ones. However, the epitopes may be altered or destroyed by fixation and permeabilization.
  5. Incubate for 15 – 60 minutes on ice in the dark. Incubation times should be optimized for best signal to noise ratio.
  6. Fill tube with Cell Staining buffer, centrifuge at 250 x g for 5 min., discard supernatant, and repeat.
  7. After the last wash discard the supernatant (there will be approximately 100 μl residual). Gently vortex to break up the pellet.
  8. Add 0.5 ml Fixation buffer and incubate for 20 min. at room temperature in the dark.
  9. Remove fixative by washing cells once as described in step 6. At this point cells can be resuspended in cell staining buffer and stored at 4°C in the dark (short term).

Permeabilization of Cells

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  1. Dilute 10X Intracellular Staining Perm Wash Buffer to 1X with DI water.
  2. Resuspend cells fixed cells in Intracellular Staining Perm Wash Buffer and centrifuge at 350 x g, RT for 5-10 minutes.
  3. Repeat wash twice.
  4. Resuspend cells in residual Intracellular Staining Perm Wash Buffer (~100 μl) by gentle vortexing to disrupt pellet. Add conjugated antibodies to intracellular antigens (isotype controls, or the combination of intracellular and surface markers) at the recommended/optimized concentration. Incubate in the dark for 20 -60 min. at RT.
  5. Wash twice in 2 ml of Intracellular Staining Perm Wash Buffer and centrifuge at at 350 x g, RT for 5-10 minutes.
  6. Resuspend in 0.5 ml Cell Staining Buffer and analyze along with the relevant controls.

1Foster, Barbara, et al. "Detection of intracellular cytokines by flow cytometry. " Current protocols in immunology (2007): 6-24.

Detection of Nuclear Proteins by Flow Cytometry

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Nuclear proteins such as transcription factors often require stronger permeabilization conditions compared to cytoplasmic proteins. This is due to differences in the composition between the nuclear and cytoplasmic membranes. In addition many nuclear proteins are bound to DNA making the antigen less accessible. The protocol below is recommended for transcription factors such as FoxP3, T-bet, and GATA-3. Please see antibody data sheet for specific buffer recommendations.

Materials

  • Cell Staining Buffer (Sony Cat. No. 2701005)
  • True-Nuclear™ Transcription Factor Buffer Set (Sony Cat. No. 2722005) Contains:
    • True-Nuclear™ 4X Fix Concentrate (30 mL)
    • True-Nuclear™ Fix Diluent (100 mL)
    • True-Nuclear™ 10X Perm Buffer (100 mL)
  • Antibodies to cell surface markers conjugated to fluorochromes (if desired)
  • Antibodies to intracellular proteins directly conjugated to fluorochromes
  • Deionized water (DI)
  • 15 or 50 mL conical centrifuge tubes
  • 5 ml flow cytometry tubes

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  1. Harvest and prepare cells according to appropriate protocol for surface staining.
  2. Centrifuge cells at 350 x g for 5 min.
  3. Discard supernatant and resuspend in an appropriate volume of Cell Staining Buffer so that the final cell concentration of cells is 5 – 10 x 106 cells/mL.
  4. Add the appropriate amount of labeled primary antibody to cell surface markers. If the primary antibody needs to be diluted use Cell Staining Buffer. Alternatively all cell surface markers can be stained with intracellular ones. However, the epitopes may be altered or destroyed by fixation and permeabilization.
  5. Incubate for 15 – 60 minutes on ice in the dark. Incubation times should also be optimized.
  6. Fill tube with Cell Staining buffer, centrifuge at 250 x g for 5 min., discard supernatant, and repeat.
  7. After the last wash discard the supernatant (there will be approximately 100 μl residual). Gently vortex to break up the pellet.
  8. Dilute the True-Nuclear™ 4X Fix Concentrate to 1 X using True-Nuclear™ Fix Diluent.
  9. Add 1 mL of Transcription Factor 1X Fix solution to each tube, vortex and incubate in the dark, RT, for 30-60 min.
  10. Dilute True-Nuclear™ 10X Perm Buffer (100 mL) to 1X using DI.
  11. Without washing add 2 mL of 1X True-Nuclear™ 1X Perm Buffer to each tube.
  12. Centrifuge tubes at 350 x g at room temperature for 5 min., discard the supernatant.
  13. Repeat steps 11 and 12. Resuspend pellet in 100 µL 1X True-Nuclear™ 1X Perm Buffer.
  14. Add the appropriate amount of fluorochrome conjugated antibodies for detection of intracellular antigen(s) (or, if appropriate antibodies to cell surface and intracellular antigens) to each tube and incubate in the dark for at least 30 minutes at RT.
  15. Centrifuge tubes at 350 x g at room temperature for 5 min., and discard the supernatant.
  16. Add 2 mL of cell staining buffer to each tube.
  17. Centrifuge tubes at 350 x g at room temperature for 5 min., and discard the supernatant.
  18. Resuspend in 0.5 mL cell staining buffer then analyse the tubes on a flow cytometer.

Detection of Phosphorylated and Other Intracellular Antigens by Flow Cytometry

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Phosphorylated proteins can be some of the most difficult to detect. Due to the transient nature of protein phosphorylation, cells should be fixed immediately after stimulation. It is often preferable to use whole blood or cultured cells for staining phosphorylated proteins. Phosphorylation is sensitive to a variety of stimuli and can be impacted by sample handling. Many phosphorylated proteins of interest are transcription factors. Antigen accessibility is challenging. As a result harsh alcohol-based permeabilization is recommended for phosphorylated proteins and some intracellular proteins.Unlike cytokine expression that may require hours of stimulation, phosphorylation occurs in minutes (10 -15 ). A time course is recommended for optimal results. The protocol below was adapted from Krutzik and Nolan, 20032. Conditions will vary by phosphoprotein. Please see antibody datasheet for more information.

Materials

  • Stimulation agent such as a cytokine or PMA/ionomycin
  • 32% formaldehyde
  • Ice cold methanol
  • Cell Staining Buffer (Sony Cat. No. 2701005)
  • Antibodies to intracellular and cell surface proteins directly conjugated to fluorochromes. It is especially important to include negative controls such as isotype controls as intracellular staining often has higher backgrounds.
  • 15 or 50 mL conical centrifuge tubes
  • 5 ml flow cytometry tubes
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  1. Stimulate cells with the appropriate agent in culture media
  2. Have fixative ready to add as simulations are short and time sensitive. Once the incubation time has ended add enough formaldehyde to bring the final concentration to 1.5% (e.g. 47 μL of 32% per mL of media).
  3. Incubate for 10 min at room temperature.
  4. Centrifuge tubes at 350 x g at room temperature for 5 min., and discard the supernatant.
  5. Resuspend cells in ~500 μl ice-cold methanol with vigorous vortexing.
  6. Incubate for 10 min. at 4°C. At this point cells can be stored for several weeks at -20°C.
  7. Fill tube with Cell Staining buffer, centrifuge at 250 x g for 5 min., discard supernatant, and repeat.
  8. Resuspend cells to a concentration of 5 – 10 X 105 cells in 100 μl Cell Staining buffer.
  9. Add the appropriate amount of fluorochrome conjugated antibodies to each tube and incubate in the dark for 15-30 minutes at RT.
  10. Fill tube with Cell Staining buffer, centrifuge at 250 x g for 5 min., and discard supernatant.
  11. Resuspend cells in 100 μl Cell Staining buffer and analyze.

2Krutzik, Peter O., and Garry P. Nolan. "Intracellular phospho‐protein staining techniques for flow cytometry: Monitoring single cell signaling events." Cytometry Part A 55.2 (2003): 61-70.

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