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 today’s researcher to accurately identify previously undetectable intracellular markers, such as cytoplasmic or nuclear enzymes, oncoproteins, cytokines, and immunoglobulins.
The FIX & PERM® cell fixation & permeabilization kit contains 2 reagents: Fixation Medium (Reagent A) and Permeabilization Medium (Reagent B). Cells in suspension are treated sequentially with Reagents A and B. This procedure gives antibodies access to intracellular structures and leaves the morphological scatter characteristics of cells intact. The optimized formulations of these reagents decrease background staining and allow the simultaneous addition of permeabilization medium and fluorophore-labeled antibodies.
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).
FIX & PERM reagent is produced for Life Technologies Laboratories by An Der Grub Bio Research GmbH, Austria.
|Peripheral blood mononuclear cells stained with FITC-conjugated mouse anti–human myeloperoxidase (MPO). Representative forward scatter (FSC) and side scatter (SSC) patterns and reaction patterns are shown.|
FIX & PERM® cell fixation & permeabilization reagents are intended for the fixation (Reagent A) and permeabilization (Reagent B) of cells in suspension. This procedure facilitates antibody access to intracellular structures and leaves the morphological scatter characteristics of the cells intact. Specific formulations reduce background staining and allow simultaneous addition of permeabilization medium and fluorophore-labeled antibodies.
|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.|
|Human peripheral blood from normal donors was stained using a combination of anti–human CD3 monoclonal antibody (clone: S4.1) for the cell surface antigens, and anti–human/mouse ZAP-70 monoclonal antibody (clone: 1E7.2), for intracellular detection. The cells were fixed and/or permeabilized using FIX & PERM® cell fixation & permeabilization reagents. The profiles represent lymphocyte gate.|
|Product||Class||Quantity||Cat. No. |
|FIX & PERM® Fixation Medium A, Bulk ||GPR* ||1 x 100 mL||GAS001S100|
|FIX & PERM® Permeabilization Medium B, Bulk ||GPR* ||1 x 100 mL||GAS002S100|
|FIX & PERM® Fixation and Permeabilization Kit ||GPR* ||1 x 5 mL||GAS003|
|FIX & PERM® Fixation and Permeabilization Kit ||GPR* ||4 x 5 mL||GAS004|
*General Purpose Reagent
|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)|
Other lysing solutions should not be used prior to the use of FIX & PERM® cell fixation & permeabilization reagents.
Protocol for Intracellular Staining With 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.
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.
Protocol for 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.
Add 0.5 mL of RPMl 1640 (without additives) to each tube to bring the volume to 1 mL.
2. 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.
3. 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.
4. Incubate for 4 hours at 37°C in a 7.5% humidified CO2 incubator.
5. At the end of the incubation period, mix the cells again and aliquot 100 µL into 12 x 75 mm snap-cap tubes.
6. 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).
7. Incubate in the dark for 15 minutes at room temperature.
8. 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.
9. 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.
10. 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.
11. Incubate for 20 minutes at room temperature.
12. Wash twice with wash medium and resuspend pellet in FACS fix.
13. Analyze by gating on SSC and FL-3 (3- or 4-color assay).
Vang KB et al. (2008) Roles of Pofut1 and O-Fucose in Mammalian Notch Signaling. J Biol Chem 283:13638–13651. (phosphor-STAT5)
LeibundGut-Landmann S et al. (2008) Stimulation of dendritic cells via the dectin-1/Syk pathway allows priming of cytotoxic T cell responses. Blood 112(13): 4971–4980.
Rosen HR et al. (2008) Pretransplantation CD56+ innate lymphocyte populations associated with severity of hepatitis C virus recurrence. Liver Transpl 14:31–40. (IFNy)
Basu S et al. (2008) Foxp3-mediated induction of Pim 2 allows human T regulatory cells to preferentially expand in rapamycin. J Immunol 180:5794–5798. (IL-2)
Libani IV (2008) Decreased differentiation of erythroid cells exacerbates ineffective erythropoiesis in β-thalassemia. Blood 112(3): 875-885. (phospho-JAK2)
El Shikh MEM et al. (2009) T-independent antibody responses to T-dependent antigens: a novel follicular dendritic cell-dependent activity. J Immunol 182: 3482-3491. (Phosphotyrosine)
Nieto WG et al. (2009) Increased frequency (12%) of circulating chronic lymphocytic leukemia–like B cell clones in healthy subjects using a highly sensitive multicolor flow cytometry approach. Blood 114(1):33–37.
Meyer zum Büschenfelde C et al. (2010) Recruitment of PKC-βII to lipid rafts mediates apoptosis-resistance in chronic lymphocytic leukemia expressing ZAP-70. Leukemia 24:141–152.