Flow Cytometry Antibodies

Flow cytometry is a powerful analytical tool which enables the high-throughput acquisition and analysis of extremely heterogeneous cell populations for interrogation and analysis at the single-cell level. As a laser-based technology, flow cytometry exploits a cell’s light scatter properties to characterize not just the size (forward light scatter) and granularity (side light scatter) of a cell, but also the fluorescence resulting from immunofluorescent staining. Fluorochrome labeled antibodies specific for cell surface markers of lineage, activation status, costimulation, and MHC gene products enable multiparameter analysis of a given cell’s phenotype. With the advent of intracellular staining methods, not just the cell surface phenotype, but also the effector function (e.g., cytokine, alarmin, defensin, granzyme production) and phospho-protein activation state can be measured on a single cell basis for a multitude of cellular biomarkers.

Aviva Systems Biology Corporation is very pleased to announce the release of over 8000 antibodies for immunofluorescent staining and flow cytometric analysis of human, mouse, and rat cellular antigens – including CDs, cytokines, transcription factors, nuclear hormone receptors, and phospho-proteins. These antibodies are validated by hundreds of publications, as well strict purity, rigorous chemistry, and meticulous QC performance specifications. Available as conjugates of more than 10 different fluorochromes, this antibody portfolio enables the truly expansive power of multi-parameter analysis at the single-cell level. As your partner in cell analysis research, Aviva Systems Biology Corporation provides these reagents at the most competitive and cost-effective pricing you’ll find, and we are happy to provide sample aliquots to help convince you of our commitment to best quality as well as best pricing.

This protocol describes the method for staining fresh, live cells by directly-labeled primary antibodies or indirectly using a fluorochrome-labeled second step reagent. This approach is useful for staining lysed whole blood, Ficoll-Hypaque-purified peripheral blood mononuclear cells, and single-cell suspensions of splenocytes, thymocytes, lymph node, and other tissue types. In addition to enabling analysis of cell lineage, this protocol can be used to staining activation markers, costimulatory molecules, and adhesion molecules.

This protocol describes the use of paraformaldehyde fixation with saponin-mediated cell permeabilization to enable staining of intracellular proteins, such as cytokines, interferons, and tumor necrosis factors. A critical consideration for success of this method is the use of a protein transport inhibitor, such as monensin or Brefeldin A, during the last 4 hours of cell activation, in order to yield sufficient accumulation of the induced target protein at the Golgi so that it may be detectable. This method is compatible for staining of cell surface receptors. For greatest utility, cell surface receptors should be stained prior to fixation *or* successful staining of the fixed epitope of the surface receptor should be pre-verified.

This protocol expands on the paraformaldehyde fixation/saponin-permeabilization method to enable staining of nuclear-associated proteins, such as transcription factors (T-bet, GATA3, FoxP3) and nuclear hormone receptors (e.g., RORgt). This protocol is compatible with staining of cell surface receptors and cytokines, in addition to transcription factors. For greatest utility, cell surface receptors should be stained prior to fixation *or* successful staining of the fixed epitope of the surface receptor should be pre-verified.

“Phospho-Flow” cytometry is a powerful technique that measures protein phosphorylation events at the single-cell level, an important feature that distinguishes this method from other antibody-based approaches to monitoring phosphorylation state, such as Western blotting or membrane-based arrays. The method allows for simultaneous analysis of multiple signaling proteins for multi-parameter analysis. This detailed protocol for flow cytometry-based screening of a given target’s phosphorylation level using phosphorylation-specific rabbit monoclonal antibodies to stain cells fixed in paraformaldehyde and methanol.

Protocol: Immunofluorescent Staining of Phosphorylated Proteins in PFA/Methanol-Fixed Cells for Flow Cytometry (aka Phospho-Flow)

“Phospho-Flow” cytometry is a powerful technique that measures protein phosphorylation events at the single-cell level, an important feature that distinguishes this method from other antibody-based approaches to monitoring phosphorylation state, such as Western blotting or membrane-based arrays. The method allows for simultaneous analysis of multiple signaling proteins for multi-parameter analysis. This detailed protocol for flow cytometry-based screening of a given target’s phosphorylation level using phosphorylation-specific rabbit monoclonal antibodies to stain cells fixed in paraformaldehyde and methanol.

Materials

  • Cells, cultured to >90% viability and 80-90% confluency
  • T75 cell culture flask (or appropriate cell culture vessel)
  • Phosphate-buffered saline
  • 0.5% trypsin solution
  • 50 mL Falcon™ tubes
  • 16% paraformaldehyde solution
  • Ice-cold 100% methanol
  • 96-well V-bottom plate
  • Staining buffer (1% BSA, PBS, 0.05% sodium azide)
  • Phospho-specific antibody
  • Isotype control antibody
  • Flow cytometer

Cell treatment and preparation

The cell treatment procedure will vary depending on the cell line and the desired treatment(s) to be used. This representative protocol is for adherent (HeLa) cells.

Cell culturing. Culture the desired cell line in the appropriate medium to>90% viability. Seed cells the day before in T75 flasks. When cells reach 80-90% confluence, aspirate media to remove.

Cell treatment. Add the appropriate medium containing the desired amount of treatment additive. Incubate cells at 37°C for the desired length of time.

Treatment conditions will vary based on the phospho target and cell type and may require optimization.

Detach cells. Aspirate the media supernatant. Wash cells by adding 10 mL of room-temperature PBS to the flask, then aspirate the PBS. Repeat for a total of two PBS washes. Add 1.5 mL of 0.5% trypsin and incubate at 37°C for 1-3 min. Detach cells from the flask by adding 10 mL of PBS and using gentle pipetting. Transfer cell suspension to 50 mL falcon tubes.

Wash cells. Pellet cells by centrifugation - 1,500xg for 5min. Aspirate the supernatant and resuspend cell pellet in 10 mL of PBS. Repeat for a total of two washes. Resuspend the final cell pellet in 2mL of PBS and count the cells.

Fix cells. Add 100 µL of 16% paraformaldehyde per 1 mL of cell suspension. Incubate cells at room temperature for 10 min.

Resuspend cells. Pellet cells by centrifugation - 1,500 x g for 5 min. Aspirate most of the supernatant, resuspending the cell pellet in the remaining 500 µL – 1 mL solution. Add ice-cold 100% methanol to achieve 2-4x10E6 cells/mL density (and >90% final methanol concentration) and vortex to mix. Incubate cells for 20 min at 4°C. Cells can be used immediately for flow cytometric analysis or stored at -20°C for up to four months.

Cell aliquot and wash. Aliquot ~1x10E5 cells/well (25µL of 4x10E6 cells/mL suspension) to a 96-well V-bottom plate. Add 200 µL of Staining buffer to each well. Pellet cells 2,000 rpm for 5 min at 4°C. Aspirate supernatant.

Primary antibody incubation. Prepare primary solutions by diluting antibodies in Staining buffer. Resuspend cells in 100 µL of antibody solution by pipetting. Incubate at 4°C for 1 hour.

The recommended dilution for each of fluorochrome-labeled phospho-specific IgG product is posted in the product page. Your experiment may require optimization by titration over multiple concentrations.

Include the following controls:

  1. Secondary only – Resuspend cells in Staining buffer only during the primary incubation step
  2. No secondary – Resuspend cells in Staining buffer only during both the primary and secondary incubation steps
  3. Isotype control – test at the same concentration as the primary antibody.

Pellet cells by centrifugation at 2,000 rpm for 5 min at 4°C. Aspirate supernatant. Wash by resuspending cells in 200 µL FACS buffer by pipetting. Repeat spin and aspirate supernatant.

Secondary antibody incubation. If your primary antibody is unlabeled, prepare the secondary antibody solution using an anti-rabbit IgG secondary antibody at the dilution recommended by the manufacturer. It may be necessary to titrate the secondary antibody to determine the optimal concentration. Resuspend cells in 100 µL of secondary antibody solution by pipetting gently. Incubate at 4°C for 30 min protected from light.

Wash. Pellet cells 2,000 rpm for 5 min at 4°C. Aspirate supernatant. Wash by resuspending cells in 200 µL Staining buffer by pipetting. Repeat spin and aspirate supernatant.

Resuspend cells and run samples. Resuspend cells in 100 µL Staining buffer by pipetting. Read cells on flow cytometer.

Protocol: Immunofluorescent Cytokine Staining of Fixed/Permeabilized Cells for Multiparameter Flow Cytometry. (aka Intracellular staining for cytokines, cytokine flow cytometry, PFA/saponin method)

This protocol describes the use of paraformaldehyde fixation with saponin-mediated cell permeabilization to enable staining of intracellular proteins, such as cytokines, interferons, and tumor necrosis factors. A critical consideration for success of this method is the use of a protein transport inhibitor, such as monensin or Brefeldin A, during the last 4 hours of cell activation, in order to yield sufficient accumulation of the induced target protein at the Golgi so that it may be detectable. This method is compatible for staining of cell surface receptors. For greatest utility, cell surface receptors should be stained prior to fixation *or* successful staining of the fixed epitope of the surface receptor should be pre-verified.

Materials Required:

  • Fixation Buffer (4% w/v Paraformaldehyde)
  • Flow Cytometry Permeabilization Buffer (10X; Cat# OOTA00781)
  • Flow Cytometry Staining Buffer (1X PBS, 2% FBS, 0.09% NaN3; Cat# OOTA00782)
  • 12x75 mm round bottom Falcon™ tubes or 96-well round bottom microtiter plates

Experimental Procedure

Cell Activation: A variety of methods have been reported to stimulate cells to elicit cytokines and investigators should optimize the kinetics and dose of mitogen or antigen used. In each case, a protein transport inhibitor, such as monensin or Brefeldin A, should be included during the last several hours of activation to cause accumulation of target protein at the Golgi apparatus.

Buffer Preparation. Prepare 4% Paraformaldehyde Fixation Buffer. Prepare 1X working solution of Permeabilization Buffer by diluting the 10X concentrate in distilled water prior to use. (Saponin-mediated permeabilization is a reversible process; washing away the Perm buffers with PBS staining buffer essentially closes up the saponin-created pores. Therefore, saponin should be included in any step for which cell permeabilization is needed.)

Experimental Procedure

Aliquot Cells. Aliquot cell samples to tubes in a volume and at a cell concentration suitable for staining.

Primary Antibody. Stain cell surface antigen(s) with the recommended optimal concentration of fluorochrome labeled antibodies.

Wash. Wash cells in 1-2 mL Stain Buffer. Centrifuge at 300-400 xg for 5 minutes at room temperature and discard supernatant. Approximately 100 µL residual volume will generally remain in the tube. Vortex tube (

Fix Cells. Fix cells by adding 100 µL of Fix Buffer and vortex (< 5 seconds). Incubate tubes in the dark at room temperature for 20-60 minutes. Wash cells in Stain Buffer. Centrifuge at 300-400 x g for 5 minutes at room temperature and discard supernatant.

Permeabilize Cells. Resuspend the cell pellet in 2 mL of 1X Permeabilization Buffer. Incubate tubes in the dark at room temperature for 5 minutes. Centrifuge samples at 300-400 x g for 5 minutes at room temperature for 5 minutes and discard the supernatant.

Stain Intracellular Targets: Resuspend the cells in 100 µL of 1X Permeabilization Buffer. Add the recommended amount of fluorochrome-labeled antibody for detection of intracellular antigen(s) to cells and incubate in the dark at room temperature for 20-60 minutes. Note: Antibodies for intracellular staining should always be prepared in 1X Permeabilization Buffer. Saponin-mediated permeabilization is a reversible process.

Wash. Add 2 mL of 1X Permeabilization Buffer to each tube. Centrifuge samples at 300-400 x g for 5 minutes at room temperature and discard the supernatant. Add

Resuspend and Analyze: Resuspend stained cells in an appropriate volume of Stain Buffer and acquire samples on a flow cytometer.

Protocol: Immunofluorescent Staining of Cell Surface Receptors and Nuclear Proteins for Flow Cytometry – (aka Foxp3 Staining)

This protocol expands on the paraformaldehyde fixation/saponin-permeabilization method to enable staining of nuclear-associated proteins, such as transcription factors (T-bet, GATA3, FoxP3) and nuclear hormone receptors (e.g., RORgt). This protocol is compatible with staining of cell surface receptors and cytokines, in addition to transcription factors. For greatest utility, cell surface receptors should be stained prior to fixation *or* successful staining of the fixed epitope of the surface receptor should be pre-verified.

Foxp3 / Transcription Factor Staining Buffer Kit (Cat #OOTA00774)

Kit Includes:

  • Foxp3 / Transcription Factor Fix/Perm Concentrate (4X; Cat #OOTA00779)
  • Foxp3 / Transcription Factor Fix/Perm Diluent (1X; Cat #OOTA00780)
  • Flow Cytometry Perm Buffer (10X; Cat #OOTA00780)

Other Materials Required:

  • Flow Cytometry Staining Buffer (1X PBS, 2% FBS, 0.09% NaN3; Cat# OOTA00782)

Buffer and Solution Preparation

1. Prepare fresh Transcription Factor Fixation/Permeabilization working solution by diluting Transcription Factor Fixation/Permeabilization Concentrate (1 part) with Transcription Factor Fixation/Permeabilization Diluent (3 parts). You will need 1 mL of the 1X Transcription Factor Fixation/Permeabilization working solution for each sample.

2. Prepare a 1X working solution of Flow Cytometry Perm Buffer by diluting the 10X concentrate with distilled water prior to use. You will need 3-5 mL of 1X Flow Cytometry Perm Buffer working solution for each sample.

Experimental Procedure

Antibody Incubation (Surface). Aliquot harvested cell samples to tubes in a volume and at a cell concentration suitable for staining. Stain cell surface antigens with the recommended optimal concentration of fluorochrome-labeled primary antibodies. Incubate for 20-30 minutes at 4°C or room temperature. Samples should be protected from light.

Wash. Wash cells with 1-2 mL Stain Buffer. Centrifuge samples at 300-400 xg at room temperature for 5 minutes, discard the supernatant. Vortex sample (Fix. Add 1 mL Transcription Factor Fixation/Permeabilization working solution to each tube and pulse-vortex (< 5 seconds). Incubate at 4°C or room temperature for 30-60 minutes in the dark. Centrifuge samples at 300-400 xg at room temperature for 5 minutes, discard the supernatant.

Wash. Wash cells with 1-2 mL Flow Cytometry Perm Buffer working solution. Centrifuge samples at 300-400 xg at room temperature for 5 minutes, discard the supernatant.

Block. Block with 2% normal mouse/rat serum by adding 2 µL directly to the cells. Incubate at room temperature for 15 minutes.

Antibody Incubation (Intracellular). Without washing, add to cells the recommended amount of fluorochrome-conjugated antibody for detection of intracellular antigen and incubate in the dark at room temperature for at least 30 minutes.

Wash. Wash cells with 1-2 mL Flow Cytometry Perm Buffer working solution. Centrifuge samples at 300-400 xg at room temperature for 5 minutes, discard the supernatant.

Wash. Wash cells with 1-2 mL Stain Buffer. Centrifuge samples at 300-400 xg at room temperature for 5 minutes, discard the supernatant.

Resuspend and Run. Resuspend stained cells in an appropriate volume of Stain Buffer and acquire data on a flow cytometer.

Protocol: Immunofluorescent Staining of Cell Surface Receptors for Multiparameter Flow Cytometry

This protocol describes the method for staining fresh, live cells by directly-labeled primary antibodies or indirectly using a fluorochrome-labeled second step reagent. This approach is useful for staining lysed whole blood, Ficoll-Hypaque-purified peripheral blood mononuclear cells, and single-cell suspensions of splenocytes, thymocytes, lymph node, and other tissue types. In addition to enabling analysis of cell lineage, this protocol can be used to staining activation markers, costimulatory molecules, and adhesion molecules.

Materials Required:

  • Flow Cytometry Staining Buffer (1X PBS, 2% FBS, 0.09% NaN3; Cat# OOTA00782)
  • 12x75 mm round bottom test tubes or 96-well round bottom microtiter plates

Experimental Procedure

Aliquot Cells. Prepare cells in a single cell suspension at a concentration of 2 x 10E7

- 2 x 10E8 cells/mL in Stain Buffer. Aliquot 50 µL of the cell sample to an individual well or tube.

Primary Antibody. Add the recommended amount of each primary antibody to the sample. The final volume of the cell sample + antibodies should not exceed 100 µL. Incubate at 4°C in the dark for 20-60 minutes.

Wash. Wash cells in 200-300 µL (for microtiter plates) or 1-2 mL (for tubes) Stain Buffer. Centrifuge at 300-400 x g for 5 minutes at room temperature and discard supernatant. Briefly vortex to dissociate the cell pellet. If no secondary reagent, skip to Resuspend/Run.

Secondary Antibody. If a second-step reagent is required for detection of unlabeled or biotinylated primary antibody, add 100 µL appropriately diluted secondary reagent to the dissociated cell pellet. Incubate at 4°C in the dark for 20-60 minutes. Wash cells in 200-300 µL (for microtiter plates) or 1-2 mL (for tubes) Stain Buffer. Centrifuge at 300-400 xg for 5 minutes at room temperature and discard supernatant. Briefly vortex to dissociate the cell pellet.

Resuspend and Run. Stained cells may be resuspended in an appropriate volume of Stain Buffer and acquired on a flow cytometer.

Quick Start Guide: Fluorochrome Selection for Multiparameter Flow Cytomety

FluorochromeAbbreviationExcitation LASERExcitation (nm)Emission (nm)
Allophycocyanin APC Red (640nm) 650 660
Allophycocyanin-Cyanine 5.5 APC/CY5. Red (640nm) 650 694
Allophycocyanin-Cyanine 7 APC/CY7 Red (640nm) 650 785
Cyanine 5 Cy5 Red (640nm) 647 665
Fluorescein isothiocyanate FITC Blue (488nm) 494 520
Pacific Blue™ PACBLU Violet (405 nm) 401 452
Peridinin chlorophyll PerCP Blue (488nm) 482 678
Peridinin chlorophyll-Cyanine 5.5 PerCP-Cy5.5 Blue (488nm) 482 678
Peridinin chlorophyll-Cyanine 7 PerCP-Cy7 Blue (488 nm), Green (532 nm), Yellow-Green (561 nm) 496 785
RedFluor™ 710 RedFluor 710 Red (633-640 nm) 633 710
R-phycoerythrin R-PE Blue (488 nm), Green (532 nm), Yellow-Green (561 nm) 496, 532, 561 578
R-phycoerythrin-Cyanine 5.5 R-PE/CY5.5 Blue (488 nm), Green (532 nm), Yellow-Green (561 nm) 565 693
R-phycoerythrin-Cyanine 7 R-PE/CY7 Blue (488 nm), Green (532 nm), Yellow-Green (561 nm) 496, 532, 561 785
R-phycoerythrin-Texas Red™ R-PE/TR Blue (488 nm), Green (532 nm), Yellow-Green (561 nm) 566 616
Spectral Red® (R-PE/Cy5) SPRD® (R-PE/Cy5) Blue (488 nm), Green (532 nm), Yellow-Green (561 nm) 496, 532, 561 667
VioletFluor™ 450 VioFluor 450 Violet (405 nm) 405 450
VioletFluor™ 500 VioFluor 500 Violet (405 nm) 405 500

Protocol: High Sensitivity Staining Procedure for Detecting Rare, Lowly-Expressed Receptors (3 steps; Indirect Amplification).

This protocol was developed for effectively staining sparsely-expressed cell surface receptors (e.g., 200/cell), such as toll-like receptors, cytokine receptors, and g-protein coupled receptors . This immunofluorescent staining method employs a three-step staining approach utilizing an amplifying biotinylated polyclonal second step reagent, combined with the brightest fluorochrome (e.g., SAv-PE, SAv-APC), as a third step to maximize the signal:noise ratio. This approach is useful for staining lowly-expressed receptors on cells within lysed whole blood, Ficoll-Hypaque-purified peripheral blood mononuclear cells, cell lines, and single-cell suspensions of splenocytes, thymocytes, lymph node, and other tissue types.

Materials Required:

Flow Cytometry Staining Buffer (1X PBS, 2% FBS, 0.09% NaN3; Cat# OOTA00782)
12x75 mm round bottom test tubes or 96-well round bottom microtiter plates

Primary antibody (Rat, Mouse, or Hamster IgG) – unlabeled.

Biotinylated Goat Anti- Mouse IgG (H+L) antibody, Human adsorbed (Cat #OASB01507) – For staining mouse IgG primary antibody used on human tissue

Biotinylated Goat Anti-Golden Syrian & Armenian Hamster IgG (H&L) antibody (Cat #OARA05203) – For staining hamster IgG primary antibody used on mouse tissue.

Biotinylated Goat Anti- Rat IgG (H+L) antibody, mouse adsorbed (Cat # OASB01929) – For staining rat IgG primary antibody used on mouse tissue.

Streptavidin-Phycoerythrin (SAv-PE). (Cat #OOTA00722). Bright fluorochrome with high signal:noise for FL2 detector.

Experimental Procedure:

Aliquot Cells. Prepare cells in a single cell suspension at a concentration of 2 x 10E7- 2 x 10E8 cells/mL in Stain Buffer. Aliquot 50 µL of the cell sample to an individual well or tube.

Primary Antibody. Add the recommended amount of each unlabeled primary antibody to the sample. The final volume of the cell sample + antibodies should not exceed 100 µL. Incubate at 4°C in the dark for 20-60 minutes.

Wash. Wash cells in 200-300 µL (for microtiter plates) or 1-2 mL (for tubes) Stain Buffer. Centrifuge at 300-400 x g for 5 minutes at room temperature and discard supernatant. Briefly vortex to dissociate the cell pellet.

Amplifying Secondary Antibody (Biotinylated). Add 100 µL appropriately-diluted biotinylated secondary reagent (biotin anti-mouse, rat, or hamster IgG) to the dissociated cell pellet. Incubate at 4°C in the dark for 20-60 minutes. Wash cells in 200-300 µL (for microtiter plates) or 1-2 mL (for tubes) Stain Buffer. Centrifuge at 300-400 xg for 5 minutes at room temperature and discard supernatant. Briefly vortex to dissociate the cell pellet.

Tertiary Fluorochrome Reagent. Add 100 µL appropriately-diluted Streptavidin-PE to the dissociated cell pellet. Incubate at 4°C in the dark for 20-60 minutes. Wash cells in 200-300 µL (for microtiter plates) or 1-2 mL (for tubes) Stain Buffer. Centrifuge at 300-400 xg for 5 minutes at room temperature and discard supernatant. Briefly vortex to dissociate the cell pellet.

Resuspend and Run. Stained cells may be resuspended in an appropriate volume of Stain Buffer and acquired on a flow cytometer.

Primary Reference:

Zola, Heddy. "Detection of cytokine receptors by flow cytometry." Current Protocols in Immunology 26, no. 1 (1998): 6-21.

References: Immunofluorescent Staining for Flow Cytometry

Adan, A., Alizada, G., Kiraz, Y., Baran, Y. and Nalbant, A., 2017. Flow cytometry: basic principles and applications. Critical reviews in biotechnology, 37(2), pp.163-176.

Burel, J.G., Qian, Y., Arlehamn, C.L., Weiskopf, D., Zapardiel-Gonzalo, J., Taplitz, R., Gilman, R.H., Saito, M., De Silva, A.D., Vijayanand, P. and Scheuermann, R.H., Alessandro Sette and Bjoern Peters. 2017. An integrated workflow to assess technical and biological variability of cell population frequencies in human peripheral blood by flow cytometry. The Journal of Immunology, 198(4), pp.1748-1758.

Donaldson, J.G., 2015. Immunofluorescence staining. Current protocols in cell biology, 69(1), pp.4-3.

McKinnon, K.M., 2018. Flow cytometry: An overview. Current protocols in immunology, 120(1), pp.5-1.

Roederer, M., 2002. Multiparameter FACS analysis. Current protocols in immunology, 49(1), pp.5-8.

References: Immunofluorescent Staining of PFA-Fixed/Saponin-Permeabilized Cells for Cytokines and Transcription Factors (aka cytokine staining, intracellular staining)

Björk, L., Fehniger, T. E., Andersson, U., & Andersson, J. (1996). Computerized assessment of production of multiple human cytokines at the single‐cell level using image analysis. Journal of leukocyte biology, 59(2), 287-295. (PFA/Saponin; Microscopy)

Björk, Lars. "Staining protocol for superantigen-induced cytokine production studied at the single-cell level." Superantigen Protocols. Humana Press, 2003. 165-184. (PFA/Saponin, IF microscopy)

Andersson, J., Abrams, J., Björk, L., Funa, K., Litton, M., Agren, K., & Andersson, U. (1994). Concomitant in vivo production of 19 different cytokines in human tonsils. Immunology, 83(1), 16. (PFA/saponin; microscopy)

Jung, Thomas, Uwe Schauer, Christoph Heusser, Christine Neumann, and Christian Rieger. "Detection of intracellular cytokines by flow cytometry." Journal of immunological methods 159, no. 1-2 (1993): 197-207. (Monensin, ICFC).

Prussin, Calman. "Cytokine flow cytometry: understanding cytokine biology at the single-cell level." Journal of clinical immunology 17.3 (1997): 195-204. (ICFC; monensin).

Ferrick, David A., Mark D. Schrenzel, Thera Mulvania, Beryl Hsieh, Walter G. Ferlin, and Heather Lepper. "Differential production of interferon-γ and interleukin-4 in response to Th1-and Th2-stimulating pathogens by γδ T cells in vivo." Nature 373, no. 6511 (1995): 255. (ICFC, Brefeldin A).

Foster, B., Prussin, C., Liu, F., Whitmire, J. K., & Whitton, J. L. (2007). Detection of intracellular cytokines by flow cytometry. Current protocols in immunology, 78(1), 6-24. (ICFC, monensin).

Prussin, Calman, and Dean D. Metcalfe. "Detection of intracytoplasmic cytokine using flow cytometry and directly conjugated anti-cytokine antibodies." Journal of Immunological Methods 188.1 (1995): 117-128. (ICFC, monensin)

Elson, Lynne H., Thomas B. Nutman, Dean D. Metcalfe, and Calman Prussin. "Flow cytometric analysis for cytokine production identifies T helper 1, T helper 2, and T helper 0 cells within the human CD4+ CD27-lymphocyte subpopulation." The Journal of Immunology 154, no. 9 (1995): 4294-4301. (ICFC).

Sano, Teruyuki, Wendy Huang, Jason A. Hall, Yi Yang, Alessandra Chen, Samuel J. Gavzy, June-Yong Lee et al. "An IL-23R/IL-22 circuit regulates epithelial serum amyloid A to promote local effector Th17 responses." Cell 163, no. 2 (2015): 381-393. (ICFC).

Ivanov, I.I., McKenzie, B.S., Zhou, L., Tadokoro, C.E., Lepelley, A., Lafaille, J.J., Cua, D.J. and Littman, D.R., 2006. The orphan nuclear receptor RORγt directs the differentiation program of proinflammatory IL-17+ T helper cells. Cell, 126(6), pp.1121-1133. (ICFC, RORgt)

Grant, J., Bourcier, K., Wallace, S., Pan, D., Conway, A., Seyfert‐Margolis, V. and Wallace, P.K., 2009. Validated protocol for FoxP3 reveals increased expression in type 1 diabetes patients. Cytometry Part B: Clinical Cytometry: The Journal of the International Society for Analytical Cytology, 76(2), pp.69-78. (Foxp3, ICFC)

Fontenot, J.D., Rasmussen, J.P., Gavin, M.A. and Rudensky, A.Y., 2005. A function for interleukin 2 in Foxp3-expressing regulatory T cells. Nature immunology, 6(11), p.1142. (ICFC, Foxp3).

Zhou, L., Lopes, J.E., Chong, M.M., Ivanov, I.I., Min, R., Victora, G.D., Shen, Y., Du, J., Rubtsov, Y.P., Rudensky, A.Y. and Ziegler, S.F., 2008. TGF-β-induced Foxp3 inhibits TH17 cell differentiation by antagonizing RORγt function. Nature, 453(7192), p.236. (ICFC, Foxp3).

Law, J.P., Hirschkorn, D.F., Owen, R.E., Biswas, H.H., Norris, P.J. and Lanteri, M.C., 2009. The importance of Foxp3 antibody and fixation/permeabilization buffer combinations in identifying CD4+ CD25+ Foxp3+ regulatory T cells. Cytometry Part A, 75(12), pp.1040-1050. (ICFC, Foxp3).

Grupillo, M., Lakomy, R., Geng, X., Styche, A., Rudert, W.A., Trucco, M. and Fan, Y., 2011. An improved intracellular staining protocol for efficient detection of nuclear proteins in YFP-expressing cells. Biotechniques, 51(6), pp.417-420. (ICFC, Foxp3).

Roncador, G., Brown, P.J., Maestre, L., Hue, S., Martínez‐Torrecuadrada, J.L., Ling, K.L., Pratap, S., Toms, C., Fox, B.C., Cerundolo, V. and Powrie, F., 2005. Analysis of FOXP3 protein expression in human CD4+ CD25+ regulatory T cells at the single‐cell level. European journal of immunology, 35(6), pp.1681-1691. (ICFC, Foxp3).

Chen, Q., Kim, Y.C., Laurence, A., Punkosdy, G.A. and Shevach, E.M., 2011. IL-2 controls the stability of Foxp3 expression in TGF-β–induced Foxp3+ T cells in vivo. The Journal of Immunology, 186(11), pp.6329-6337. (ICFC, Foxp3).

Santegoets, S.J., Dijkgraaf, E.M., Battaglia, A., Beckhove, P., Britten, C.M., Gallimore, A., Godkin, A., Gouttefangeas, C., de Gruijl, T.D., Koenen, H.J. and Scheffold, A., 2015. Monitoring regulatory T cells in clinical samples: consensus on an essential marker set and gating strategy for regulatory T cell analysis by flow cytometry. Cancer Immunology, Immunotherapy, 64(10), pp.1271-1286. (ICFC, Foxp3).

Korn, T., Mitsdoerffer, M., Croxford, A.L., Awasthi, A., Dardalhon, V.A., Galileos, G., Vollmar, P., Stritesky, G.L., Kaplan, M.H., Waisman, A. and Kuchroo, V.K., 2008. IL-6 controls Th17 immunity in vivo by inhibiting the conversion of conventional T cells into Foxp3+ regulatory T cells. Proceedings of the National Academy of Sciences, 105(47), pp.18460-18465. (ICFC, Foxp3).

Jäger, A., Dardalhon, V., Sobel, R.A., Bettelli, E. and Kuchroo, V.K., 2009. Th1, Th17, and Th9 effector cells induce experimental autoimmune encephalomyelitis with different pathological phenotypes. The Journal of Immunology, 183(11), pp.7169-7177. (ICFC).

Lee, Y., Awasthi, A., Yosef, N., Quintana, F.J., Xiao, S., Peters, A., Wu, C., Kleinewietfeld, M., Kunder, S., Hafler, D.A. and Sobel, R.A., 2012. Induction and molecular signature of pathogenic TH17 cells. Nature immunology, 13(10), p.991. (ICFC).

Korn, T., Bettelli, E., Gao, W., Awasthi, A., Jäger, A., Strom, T.B., Oukka, M. and Kuchroo, V.K., 2007. IL-21 initiates an alternative pathway to induce proinflammatory TH17 cells. Nature, 448(7152), p.484. (ICFC).

Maecker, H.T. "Cytokine flow cytometry: a multiparametric approach for assessing cellular immune responses to viral antigens." Clinical Immunology 110, no. 3 (2004): 222-231. (ICFC).

Freer, Giulia, and Laura Rindi. "Intracellular cytokine detection by fluorescence-activated flow cytometry: basic principles and recent advances." Methods 61, no. 1 (2013): 30-38. (ICFC).

References: Immunofluorescent Staining of Phosphorylated Proteins in Methanol-Fixed/Permeabilized Cells for Flow Cytometry – aka “Phospho-Flow”

Davies, Richard, et al. "An optimized multiplex flow cytometry protocol for the analysis of intracellular signaling in peripheral blood mononuclear cells." Journal of Immunological Methods 436 (2016): 58-63. (Phospho FC, MeTOH).

Krutzik, Peter O., and Garry P. Nolan. "Intracellular phospho‐protein staining techniques for flow cytometry: Monitoring single cell signaling events." Cytometry Part A: the journal of the International Society for Analytical Cytology 55.2 (2003): 61-70. (Phospho FC, MeTOH).

Irish, J.M., Hovland, R., Krutzik, P.O., Perez, O.D., Bruserud, Ø., Gjertsen, B.T. and Nolan, G.P., 2004. Single cell profiling of potentiated phospho-protein networks in cancer cells. Cell, 118(2), pp.217-228. (Phospho FC, MeTOH).

Krutzik, Peter O., Jonathan M. Irish, Garry P. Nolan, and Omar D. Perez. "Analysis of protein phosphorylation and cellular signaling events by flow cytometry: techniques and clinical applications." Clinical immunology 110, no. 3 (2004): 206-221. (Phospho FC, MeTOH).

Perez, O.D. and Nolan, G.P., 2006. Phospho‐proteomic immune analysis by flow cytometry: from mechanism to translational medicine at the single‐cell level. Immunological Reviews, 210(1), pp.208-228. (Phospho FC, MeTOH).

Immunofluorescent Staining for Flow Cytometry and Intracellular Staining Frequently-Asked Questions (FAQs): Question & Answers

 

Question: What is the best starting point for titrating my fluorochrome-labeled antibody and determining optimal cell staining conditions for FACS analysis?

Answer: A good starting point is to define the staining matrix as 10E5 – 10E6 cells stained in a 100ul staining volume, assuming 30min at 4C in PBS/FBS/NaN3. Within this staining volume, the following starting points are recommended, based on the fluorochrome or biotin: FITC, Biotin, PerCP: Titrate down from 1.0 ug/10E6 cells in 100ul staining volume PE, APC, PE/Cy5, PE/Cy7: Titrate down from 0.25 ug/10E6 cells in 100ul staining volume.

Question: For immunofluorescent cell staining with FACS analysis, it is strategically important to use your brightest fluorochrome for the markers which are expressed the least. Which are the brightest fluorochromes and which are the weakest?

Answer: PE, PE/Cy5, and APC are amongst the brightest fluorochromes for immunofluorescent cell staining with FACS analysis. FITC and PerCP are amongst the dimmer fluorochromes. For additional tips on this, see the methods papers in the reference section.

Question: The cell surface receptor which I want to stain for FACS analysis is very sparsely-expressed on the cell surface. What is the best approach for staining very sparsely-expressed cell surface receptors for FACS analysis?

Answer: One effective approach for amplifying signal for FACS analysis (described by Heddy Zola in Currrent Protocols in Immunology) is the use of an intermediate amplification step, yielding a 3-step staining approach. First, stain with unlabeled primary antibody, then, stain with a clean, amplifying biotinylated polyclonal anti-IgG, and finally stain with SAv-PE or SAv-APC. For additional tips on this, see the methods papers in the reference section. Zola, Heddy. "Detection of cytokine receptors by flow cytometry." Current Protocols in Immunology 26, no. 1 (1998): 6-21.

Question: Do I need to block Fc receptors prior to immunofluorescent staining for flow cytometric analysis ?

Answer: Cultured, activated macrophages, B cells, and dendritic cells are notable for upregulation of Fc receptors and subsequent “stickiness” for rodent and rabbit IgG. Treatment of such cells with a blocking anti-FcR (CD16/32), Fc fragments, or normal rodent serum matching the reagents of your experiment will help to minimize background staining and overcome stickiness of the cells. For additional tips on this, see the methods papers in the reference section.

Question: Do Fc receptors bind common fluorochromes and cause high background for cell staining and flow cytometry?

Answer: R-phycoerythrin and R-PE tandem dyes have been reported to bind FcR’s of activated B cells and macrophages, so a blocking strategy can be important. Takizawa, Fumiyoshi, Jean-Pierre Kinet, and Martin Adamczewski. "Binding of phycoerythrin and its conjugates to murine low affinity receptors for immunoglobulin G." Journal of immunological methods 162, no. 2 (1993): 269-272.

Question: For cell staining with FACS analysis, what common fluorescent dyes are compatible with the blue (488nm) laser?

Answer: The following common fluorescent dyes are compatible with the blue (488nm) laser: FITC, PE, PE/Cy5 (Spectral Red™), PerCP, PerCP/Cy5, PerCP/Cy7. For additional tips on this, see the methods papers in the reference section.

Question: For cell staining with FACS analysis, what common fluorescent dyes are compatible with the red (640nm) laser?

Answer: The following common fluorescent dyes are compatible with the red (640nm) laser: APC, APC/Cy5.5, APC/Cy7, Cy5.

Question: For cell staining with FACS analysis, what common fluorescent dyes are compatible with the violet (405nm) laser?

Answer: The following common fluorescent dyes are compatible with the violet (405nm) laser: PACBLUE, VioFluor450, VioFluor500.

Question: I am setting up cell cultures for activation and subsequent intracellular cytokine staining for FACS analysis. Do I need to include a protein transport inhibitor? If so, for how long?

Answer: For intracellular staining and FACS analysis, it is critically-important to include a protein transport inhibitor (e.g., monensin, brefeldin A) during the last 4-12 hours of cell activation in order to cause accumulation of the target at the Golgi and subsequently enhance the signal sufficiently for staining. For additional tips on this, see the methods papers in the reference section. Foster, B., Prussin, C., Liu, F., Whitmire, J. K., & Whitton, J. L. (2007). Detection of intracellular cytokines by flow cytometry. Current Protocols in Immunology, 78(1), 6-24. (ICFC, monensin).

Question: I am staining human PBMCs for Foxp3 and IL-17A and IFNgamma. Is the cell fixation/permeabilization method recommended for staining Foxp3 also compatible with staining IL-17A and IFNg?

Answer: Yes, the Foxp3 Fix/Perm buffers yield fixation conditions which are compatible for both cytokines and transcription factors (e.g., T-bet, GATA3, etc). For additional tips on this, see the methods papers in the reference section. Law, J.P., Hirschkorn, D.F., Owen, R.E., Biswas, H.H., Norris, P.J. and Lanteri, M.C., 2009. The importance of Foxp3 antibody and fixation/permeabilization buffer combinations in identifying CD4+ CD25+ Foxp3+ regulatory T cells. Cytometry Part A, 75(12), pp.1040-1050. (ICFC, Foxp3). Grupillo, M., Lakomy, R., Geng, X., Styche, A., Rudert, W.A., Trucco, M. and Fan, Y., 2011. An improved intracellular staining protocol for efficient detection of nuclear proteins in YFP-expressing cells. Biotechniques, 51(6), pp.417-420. (ICFC, Foxp3).