FIX & PERM Cell Fixation & Cell Permeabilization Kit

The FIX & PERM Cell Fixation and Cell Permeabilization Kit achieves mild fixation and permeabilization of cells that leaves their morphological scatter characteristics intact, enabling researchers to accurately identify previously undetectable intracellular markers, such as cytoplasmic or nuclear enzymes, oncoproteins, cytokines, and immunoglobulins.

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About FIX & PERM Cell Fixation and Permeabilization Kits

  • Compatible with analysis of most cellular antigens
  • No effect on cellular morphological scatter
  • Reduced background staining
  • Proven protocols

The FIX & PERM Cell Permeabilization Kit consists of matched Fixation Reagent (Medium A) and Permeabilization Reagent (Medium B) for simultaneous analysis of intracellular and cell-surface antigens in the same cell population (Figure 1). This procedure facilitates antibody access to intracellular structures and leaves the morphological light-scattering characteristics of the cells intact. These formulations reduce background staining and allow simultaneous addition of permeabilization medium and fluorophore-labeled antibodies. The Fixation Medium and Permeabilization Medium are available separately as well.

FIX & PERM reagents are suitable for the analysis by flow cytometry of normal and malignant leukocyte populations derived from various human biological samples (blood, bone marrow, and others) (Figure 2).

four different flow cytometry scatter plots showing various populations distinguishable using TNF-a, IFN, and CD4 markers

Figure 1. Use of FIX & PERM Cell Permeabilization Kit for simultaneous surface antigen and intracellular antigen staining. C57BL/6 splenocytes were left unstimulated or stimulated for 5 hours with phorbol myristate acetate (PMA) and ionomycin in the presence of brefeldin A. Cells were then surface-stained with fluorescein (FITC)-conjugated anti–mouse CD4 antibody. This step was followed by fixation and permeabilization of the sample using the FIX & PERM Cell Permeabilization Kit. Intracellular staining was performed during the permeabilization step using PE-Cy7 tandem–conjugated anti–mouse γ-interferon (γ-IFN) antibody and Pacific Blue dye–conjugated anti–mouse tumor necrosis factor α (TNF-α) antibody. Data were collected using the Attune Flow Cytometer (blue/violet) with 488 nm excitation and a 530/30 nm bandpass emission filter to detect FITC fluorescence and a 640 nm longpass filter to detect PE-Cy7 tandem fluorescence. Pacific Blue conjugate fluorescence was detected using 405 nm excitation and a 450/40 nm bandpass emission filter. (Top row) γ-IFN and TNF-α antibody co-staining of total mouse splenocytes, gated on lymphocytes, that were left unstimulated (left) or stimulated (right) with PMA and ionomycin in the presence of brefeldin A. (Bottom row) CD4+ T cell expression of TNF-α (left) and γ-IFN (right) after stimulation as described above.

Immunophenotyping of a whole-blood sample

Figure 2. Immunophenotyping of a whole-blood sample. Two-day-old whole blood was lysed with ammonium chloride. Cells were first stained with CD13-APC conjugate. Cells were washed, stained with LIVE/DEAD Fixable Near-IR dead cell stain, washed, and fixed with FIX & PERM Reagent A. Cells were then washed, permeabilized with FIX & PERM Reagent B, stained with MPO-FITC conjugate, and washed. Cells were analyzed on a BD™ LSRII flow cytometer. Gating on live cells (generally recommended, to eliminate dead cells) was performed. Further subgating is recommended in order to obtain the most accurate results.

Markers and intracellular proteins

The FIX & PERM Cell Fixation & Permeabilization Kit allows efficient detection of a wide variety of markers and intracellular proteins, including, but not limited to:

Lysosomal proteinsElastase, lactoferrin, lysozyme, myeloperoxidase, proteinase-3
Cytoplasmic CD moleculesCD3, CD13, CD22, CD62P, CD63, CD68, CD79a
Nuclear proliferation markersBrdU, Ki-67, PCNA, nuclear enzymes, TdT
OncoproteinsBcl-2, c-Myc, p53, HIV antigens, p24
Cytokines & chemokines (human)
IFN-γ, TNF-α, IL1-β, IL-2, IL-4, IL-10, IL-12, IL-13, IL-16, RANTES
Cytokines & chemokines (mouse)
IFN-γ, TNF-α, IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, IL-16
Immunoglobulins (human)
IgA, IgG, IgD, IgM, kappa, lambda
Immunoglobulins (mouse)IgG, IgM
Other moleculesZAP-70 (zeta-associated protein 70), MHC class II, phosphotyrosine, thrombospondin, cyclins, transfected cells, MDR (multidrug resistance)

Permeabilization and staining protocol

FIX & PERM cell fixation & permeabilization reagents are designed for use with all commercially available flow cytometers. This standard procedure for intracellular staining with FIX & PERM fixation & permeabilization reagents gives optimal results for most antigens. A modification using precooled absolute methanol has been shown to give better results for certain cell cycle antigens such as Ki-67 and PCNA when using FITC-conjugated antibodies. The methanol step is not recommended when using RPE-conjugated antibodies. These staining protocols are intended for use directly with the cell suspension to be analyzed.

Other lysing solutions should not be used prior to the use of FIX & PERM cell fixation & permeabilization reagents.

Intracellular staining using the FIX & PERM Cell Fixation & Permeabilization Kit
  1. For each sample to be analyzed, add the appropriate volume of the conjugated antibody directed to the cell surface marker(s) of interest, and/or the appropriate isotype control(s), to an appropriate 5 mL, 12 x 75 mm tube.
  2. Pipette 100 μL of whole blood or adjusted cells (stimulated PBMCs, bone marrow, spleen, thymus), equivalent to 1 x 106 cells, into each tube containing the conjugated antibody or isotype control.
  3. Vortex each tube gently to mix, and incubate for 15 minutes in the dark at room temperature.
  4. Add 100 μL of Reagent A (Fixation Medium) and incubate for 15 minutes in the dark at room temperature.
  5. Wash once with 3 mL wash medium (PBS + 0.1% NaN3 + 5% FBS).
  6. Centrifuge for 5 minutes at 300–350 x g, aspirate supernatant, and vortex to fully resuspend the cell pellet.
  7. Add 100 μL of Reagent B (Permeabilization Medium) and the recommended volume of intracellular antibodies or the corresponding isotype control(s).
  8. Vortex 1–2 seconds and incubate for 20 minutes in the dark at room temperature.
  9. Wash once with 3 mL wash medium (PBS + 0.1% NaN3 + 5% FBS).
  10. Centrifuge for 5 minutes at 300–350 x g and aspirate the supernatant.
  11. Resuspend cells in sheath fluid for immediate analysis, or fix in 0.5 mL of 0.1% paraformaldehyde and store at 2–8°C in the dark. Fixed cells should be analyzed within 18 hours.
Methanol modification

The methanol modification procedure is recommended for cell cycle antigens such as BrdU, Ki-67, and PCNA when using FITC-conjugated antibodies. It is not recommended when using RPE-conjugated antibodies.

  1. For each sample to be analyzed, add 100 μL of adjusted cell volume (equivalent to 1 x 106 cells) to an appropriate 5 mL, 12 x 75 mm tube.
  2. Add 100 μL of Reagent A (Fixation Medium) and incubate for 2–3 minutes at room temperature.
  3. Add 4 mL of precooled (0–4°C) absolute methanol and vortex.
  4. Incubate for an additional 10 minutes at 0–4°C.
  5. Centrifuge for 5 minutes at 300–350 x g and wash with wash medium (PBS + 0.1% NaN3 + 5% FBS).
  6. Add 100 μL of Reagent B (Permeabilization Medium) and an appropriate volume of intracellular antibodies or the corresponding isotype control(s).
  7. Vortex at low speed for 1–2 seconds and incubate for 30 minutes at room temperature.
  8. Wash once with 3 mL wash medium (PBS + 0.1% NaN3 + 5% FBS).
  9. Centrifuge for 5 minutes at 300–350 x g and aspirate the supernatant.
  10. Resuspend cells in sheath fluid for immediate analysis, or fix in 0.5 mL of 0.1% paraformaldehyde and store at 2–8°C in the dark. Fixed cells should be analyzed within 18 hours.
Stimulation of human whole blood and intracellular cytokine staining with FIX & PERM cell fixation & permeabilization reagents

VERY IMPORTANT: Blood must be collected into heparinized tubes.

  1. Dispense 0.5 mL of heparinized whole blood into each of two 12 x 75 mm snap-cap tubes.
  2. Add 0.5 mL of RPMl 1640 (without additives) to each tube to bring the volume to 1 mL.
  3. To the first tube, add 10 µg brefeldin A (20 µL of a stock solution of brefeldin A at a concentration of 0.5 mg/mL in 100% EtOH and stored at –20°C). Mix contents of the tube gently. This tube represents the resting cell population.
  4. To the second tube, add 10 µg brefeldin A, 25 ng PMA (2.5 µL of a stock solution of PMA at a concentration of 1 mg/mL in DMSO and diluted 1:100 in RPMI 1640 (without additives) and stored at –20°C), and 1 µg ionomycin (1 µL of a stock solution of ionomycin at a concentration of 1 mg/mL in DMSO and stored at –20°C). Mix contents of the tube gently. This tube represents the activated cell population.
  5. Incubate for 4 hours at 37°C in a 7.5% humidified CO2 incubator.
  6. At the end of the incubation period, mix the cells again and aliquot 100 µL into 12 x 75 mm snap-cap tubes.
  7. Add 5 µL of the appropriate antibodies for phenotyping cells (cell-surface staining) (e.g., CD3-TRI-COLOR, CD4-TC, CD8-TC, CD45-TC, or CD69-TC for a three-color staining assay; either FITC or RPE may be appropriate for a two-color assay).
  8. Incubate in the dark for 15 minutes at room temperature.
  9. Add 100 µL of Reagent A from the FIX & PERM Kit. Mix cells gently and incubate for an additional 15 minutes at room temperature in the dark.
  10. Wash twice with wash medium (PBS + 1 % BSA, 0.1 % NaN3, 1 % FBS). The pellet becomes less cohesive on the second wash, so use caution to avoid losing cells.
  11. To the washed cells add 100 µL of Reagent B from the FIX & PERM Kit and 1–10  µL of each anti-cytokine antibody OR isotype control.
  12. Incubate for 20 minutes at room temperature.
  13. Wash twice with wash medium and resuspend pellet in FACS fix.
  14. Analyze by gating on SSC and FL-3 (3- or 4-color assay).

References

Resources

Fluorophore and reagent selection guide for flow cytometry

Download Flow Cytometry Protocols Handbook

Spectral Flow Cytometry Fundamentals

Invitrogen eBioscience Resources—Selection guides, Best Protocols, product performance and more.

Intracellular Staining for Flow Cytometry How-To Video—for detecting cytokines and intranuclear markers.

Flow Cytometry Learning Center—Access flow cytometry educational resources for better experiment planning and execution.

Flow Cytometry Panel Builder—Design your flow cytometry panel with this online tool for a simplified, customizable experience to fit your needs.

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