Fix perm cell fixation permeabilization kit

Manufactured by Thermo Fisher Scientific

The FIX & PERM Cell Fixation & Permeabilization Kit is a laboratory tool designed to prepare cell samples for analysis. It is used to fix and permeabilize cells, enabling the detection of intracellular targets. The kit provides reagents and a standardized protocol to achieve consistent sample preparation.

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FIX & PERM Cell Fixation & Cell Permeabilization Kit Fix and Perm Cell fixation and cell permeabilization Kit Fix&Perm Cell Fixation and Cell Permeabilization Kit FIX/PERM Cell fixation and permeabilization kit FIX & PERM Cell Permeabilization Kit Cell Fixation/Permeabilization Kit Fix/Perm kit Fix/Permeabilization kit Fixation/permeabilization kit Fix/Perm

4 protocols using fix perm cell fixation permeabilization kit

Multiparametric flow cytometry analysis of T cell activation

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T cells from various experiment groups: T₀, T_NPP12 and T_PP12 were cultured in complete RPMI medium, or with T2 cell (pulse with synthetic peptide pool for 2 h on rotator), or with peptide pool for 4 h, followed by adding GolgiPlug (Biolegend, San Diego, CA). To exclude dead cells, samples were stained with Live/Dead dye (Biolegend, San Diego, CA), followed by surface staining (fluorochrome-conjugated antibodies: anti-CD3, anti-CD4, and anti-CD8, Biolegend, San Diego, CA) for 30 min on ice. Cells were then fixed with Fix & Perm Cell Fixation & Permeabilization Kit (ThermoFisher) according to the instruction, stained with fluorochrome-conjugated anti-IFN-γ and anti-IL-2 (Biolegend, San Diego, CA) for 30 min on ice, washed and resuspended in staining buffer with 1% PFA, followed by flow cytometry analysis by BD LSR Fortessa (Beckman Dickinson, Franklin Lakes, NJ.). Flow cytometry data was analyzed by FlowJo (Three Star, Ashland, OR).

Miyaguchi K., Wang H., Black K.L., Shiao S.L., Wang R, & Yu J.S. (2023). Activated T cell therapy targeting glioblastoma cancer stem cells. Scientific Reports, 13, 196.

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Interferon-Alpha and IL-22 Modulate HUVEC Survival

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HUVECs (ATCC, CRL-1730) were cultured in growth media (EGM-2 Endothelial Cell Growth Medium-2 BulletKit, Lonza) containing growth factors IGF1, FGF2, EGF, and VEGFA according to the manufacturer’s instructions, unless otherwise stated. Cells (5 × 10³ per 96-well) were stimulated with 1,000 U/mL human IFN-α (1100-1, PBL Assay Science), and/or 100 ng/mL IL-22 (200-22, PeproTech) for 8 hours for gene expression analyses or for 5 days for survival and proliferation analyses. For survival experiments, cells were stained with SYTO 13/SytoX ORANGE (S34854, S11368, Invitrogen) and analyzed by flow cytometry. Live cells were counted as total Syto single-positive cells per condition. For proliferation experiments, cells were fixed, permeabilized (FIX & PERM Cell Fixation & Permeabilization Kit, Thermo Fisher Scientific), stained for KI67, and analyzed by flow cytometry.

Mylonas A., Hawerkamp H.C., Wang Y., Chen J., Messina F., Demaria O., Meller S., Homey B., Di Domizio J., Mazzolai L., Hovnanian A., Gilliet M, & Conrad C. (2023). Type I IFNs link skin-associated dysbiotic commensal bacteria to pathogenic inflammation and angiogenesis in rosacea. JCI Insight, 8(4), e151846.

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Immunohistochemistry of Colonic RORγt+ Cells

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Colons were fixed with 4% paraformaldehyde (Merck) in PBS under agitation at 4°C. Fixed colons were further washed with PBS containing 1% Triton X-100 (Sigma) and 2% FCS (Sigma) overnight at 4°C. Samples were further processed with FIX&PERM cell fixation & permeabilization kit (Thermo Fischer) for RORγt staining. Briefly, samples were incubated with 1x fixation/permeabilization buffer overnight. After washing with permeabilization buffer, samples were stained with rat α-RORγt (AFKJS-9) Ab (eBioscience) for 2 hr at 4°C. Samples were then stained with biotin conjugated α-rat secondary Ab overnight after washing with permeabilization buffer. Finally, samples were incubated with Cy3-conjugated streptavidin (Jackson ImmunoResearch) and AF647-conjugated α-B220 (RA3-6B2) Ab (Biolegend) for 2 hr. Podoplanin was detected with 8.1.1 Ab (Biolegend). Microscopy analysis was performed using a confocal microscope (Zeiss LSM-710) and images were processed with ZEN 2010 software (Carl Zeiss) and Imaris (Bitplane).

Emgård J., Kammoun H., García-Cassani B., Chesné J., Parigi S.M., Jacob J.M., Cheng H.W., Evren E., Das S., Czarnewski P., Sleiers N., Melo-Gonzalez F., Kvedaraite E., Svensson M., Scandella E., Hepworth M.R., Huber S., Ludewig B., Peduto L., Villablanca E.J., Veiga-Fernandes H., Pereira J.P., Flavell R.A, & Willinger T. (2018). Oxysterol Sensing through the Receptor GPR183 Promotes the Lymphoid-Tissue-Inducing Function of Innate Lymphoid Cells and Colonic Inflammation. Immunity, 48(1), 120-132.e8.

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T Cell Functional Characterization by Flow Cytometry

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T cell functions were assessed using flow cytometry as described previously (14 (link)). Briefly, 6C5 T cells were incubated with T2 cells ± peptide (10 μg/ml) in the presence of GolgiStop (0.7 μl/ml; BD Biosciences) and anti-CD107a–FITC (clone H4A3; BD Biosciences). Negative control tubes contained equivalent DMSO or no T2 cells, and positive control tubes contained PMA (10 ng/ml; Sigma-Aldrich) and ionomycin (1.5 μg/ml; Sigma-Aldrich). Cells were incubated for 6 hr at 37°C, washed with FACS buffer (Thermo Fisher Scientific), and stained for 15 min at 4°C with anti-CD8a–PerCP-Cy5.5 (clone SK1), anti-CD19–BV510 (clone HIB19), and Zombie NIR (all from BioLegend). After a further wash, cells were fixed/permeabilized using a FIX & PERM Cell Fixation & Permeabilization Kit (Thermo Fisher Scientific), washed again, and stained for 20 min at 4°C with anti-IFN-γ–V450 (clone B27; BD Biosciences), anti-IL-2–APC (clone MQ1-17H12; BioLegend), anti-MIP-1β–PE (clone D21-1351; BD Biosciences), and anti-TNF-α–PE-Cy7 (clone MAb11; BioLegend). Data were acquired immediately using a FACSCanto flow cytometer (BD Biosciences) and analyzed using FlowJo software version 9.9.4 (FlowJo LLC). The gating strategy is shown in Supplemental Fig. 1.

Man S., Redman J.E., Cross D., Cole D.K., Can I., Davies B., Hashimdeen S.S., Reid R., Llewellyn-Lacey S., Miners K.L., Ladell K., Lissina A., Brown P.E., Wooldridge L., Price D.A, & Rizkallah P.J. (2021). Synthetic peptides with inadvertent chemical modifications can activate potentially autoreactive T cells. Journal of immunology (Baltimore, Md. : 1950), 207(4), 1009-1017.

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