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-Cy®7 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® Acoustic Focusing 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-Cy®7 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.

flow cytometry scatter plot showing granulocyte, monocyte, and lymphocyte populations via side scatter analysis and live-cell staining with a second scatter plot of just the lymphocytes showing three subpopulations distinguished by CD13 and MPO staining

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 proteins Elastase, lactoferrin, lysozyme, myeloperoxidase, proteinase-3
Cytoplasmic CD molecules CD3, CD13, CD22, CD62P, CD63, CD68, CD79a
Nuclear proliferation markers BrdU, Ki-67, PCNA, nuclear enzymes, TdT
Oncoproteins Bcl-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 molecules ZAP-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.
  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

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

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FIX & PERM reagent is produced for Thermo Fisher Scientific laboratories by An Der Grub Bio Research GmbH, Austria.

For Research Use Only. Not for use in diagnostic procedures.