Facs perm 2

Manufactured by BD

Sourced in United States

The BD FACS Perm II is a laboratory equipment designed for cell fixation and permeabilization. It is used to prepare samples for flow cytometry analysis by allowing the introduction of antibodies or other reagents into the cells. The device facilitates the measurement of intracellular proteins, cytokines, and other cellular components.

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Lab products found in correlation

Facs lyse, by BD (12 mentions) Cisplatin, by Enzo Life Sciences (6 mentions) Iridium intercalator, by Standard BioTools (3 mentions) Fluorescein isothiocyanate (fitc), by BD (2 mentions) Brefeldin a, by Merck Group (2 mentions) Ionomycin, by Thermo Fisher Scientific (2 mentions) Monensin, by Thermo Fisher Scientific (2 mentions) Golgistop, by BD (2 mentions) 96 well deep well plates, by Merck Group (2 mentions) Maxpar x8 labeling kit, by Standard BioTools (2 mentions) Helios mass cytometer, by Standard BioTools (2 mentions) Maxpar 8 labeling kits, by Standard BioTools (2 mentions) Cd8 apc cy7, by BD (1 mentions) Ifn γ pacific blue, by BioLegend (1 mentions) Il 17 af488, by BioLegend (1 mentions) Anti il 2 apc, by BD (1 mentions) Facscalibur flow cytometry, by BD (1 mentions) Countbright absolute counting beads, by Thermo Fisher Scientific (1 mentions) Optilyse c, by Beckman Coulter (1 mentions) Aqua dead cell stain kit, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

FACS Perm (5 citations) BD FACS II (3 citations) BD FACSTM (2 citations) BD FACS Perm II Buffer (2 citations)

17 protocols using facs perm 2

Intracellular Cytokine and CD25+CD134+ Assays

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Intracellular cytokine assays were performed as previously described [14 (link),26 (link)]. Briefly 0.5 mL fresh sodium heparin anticoagulated blood was incubated with SEB or antigens (as above) for 2 h at 37 °C, before the addition of 10 µg/mL brefeldin A (Sigma-Aldrich), and incubated for a further 4 h, before addition of 2 mM EDTA. Aliquots were stained with CD3-PerCP-Cy5.5, CD4-PE-Cy7, CD8-APC-Cy7 and CD73-PE, lysed with FACSLyse (BD Biosciences), permeabilized with FACSPerm2 (BD Biosciences), and then incubated with anti-IL-2-APC (BD Biosciences), IFN-γ-Pacific Blue (BioLegend, San Diego, CA, USA) or –FITC (BD Biosciences) and IL-17-AF488 (BioLegend). Cells were then analyzed on an LSR II, as described above.
CD25+CD134+ (“OX40”) assays were performed as previously described [33 (link)]. Briefly, 0.25 mL IMDM was added to 0.25 mL fresh Na Hep anticoagulated blood and incubated with SEB or antigens (as above) for 40–48 h at 37 °C. Aliquots were stained with CD3-PerCP-Cy5.5, CD4-PE-Cy7, CD25-APC, CD134-FITC and CD73-PE, lysed with Optilyse C, and then analyzed on an LSR II, as described above.

Seddiki N., Zaunders J., Phetsouphanh C., Brezar V., Xu Y., McGuire H.M., Bailey M., McBride K., Hey-Cunningham W., Munier C.M., Cook L., Kent S., Lloyd A., Cameron B., Fazekas de St Groth B., Koelsch K., Danta M., Hocini H., Levy Y, & Kelleher A.D. (2021). CD73+ CD127high Long-Term Memory CD4 T Cells Are Highly Proliferative in Response to Recall Antigens and Are Early Targets in HIV-1 Infection. International Journal of Molecular Sciences, 22(2), 912.

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Characterization of IL-17 Producing T Cells

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The PBMCs or CD4⁺ T cells were re-suspended to approximately 2 × 10⁶ cells in 100 μL ice-cold PBS. Fluorescein isothiocyanate (FITC)-labeled anti-human CD4 antibody (Biolegend) was added to each tube along with Phycoerythrin-Cy7 (PE-CY7) anti-human CD45RO antibody (BD biosciences). The cells were incubated for 30 min at 4°C in the dark. After washing with PBS, these cells were stimulated with 1 μg/mL ionomycin, 20 ng/mL phorbol myristate acetate (PMA), and 2 μmol/L monensin (all from eBioscience) for 5 h under a 5% CO₂ /37°C environment in a 24-hole culture plate (Costar). After 5 h, cells were harvested and washed with PBS. They were fixed and permeabilized by FACS™ Perm 2 (BD Bioscience) according to the manufacturer's instructions. For intracellular staining, the cells were stained with phycoerythrin (PE)-labeled anti-human IL-17 antibody (BD biosciences) for 30 min. After washing with PBS, these cells were re-suspended with 200 μL PBS in tube, respectively. After adding 10 μl CountBright™ Absolute Counting Beads (Thermo Fisher) into each tube, samples were measured by FACS calibur flow cytometry (BD Biosciences).

Long Q., Liao Y.H., Xie Y., Liang W., Cheng X., Yuan J, & Yu M. (2016). Coxsackievirus B3 Directly Induced Th17 Cell Differentiation by Inhibiting Nup98 Expression in Patients with Acute Viral Myocarditis. Frontiers in Cellular and Infection Microbiology, 6, 171.

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Multicolor Flow Cytometry Analysis of Blood

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An amount of 100 µL of fresh Na heparin anti-coagulated peripheral blood samples (<4 h after venesection) were stained with monoclonal antibodies (mAb) according to the manufacturers’ directions, incubated for 15 min at RT, lysed with Optilyse C (Beckman Coulter, Brea, CA, USA) for 10 min at RT, washed with 2 mL PBS and resuspended in 250 µL 0.5% paraformaldehyde in PBS, as previously described [12 (link),37 (link)]. Intracellular staining for cytotoxic granules and granzymes was performed using FACSLyse (BD Biosciences, San Jose, CA, USA) and FACSPerm 2 (BD Biosciences) as previously described [12 (link)]. The mAb’s used in this study are listed in Supplementary Table S3. Fixed samples were kept at 4 °C before 14-colour analysis, within 24 h, on an LSR II (BD Biosciences) as previously described [12 (link),37 (link),38 (link)].

Frith K., Munier C.M., Hastings L., Mowat D., Wilson M., Seddiki N., Macintosh R., Kelleher A.D., Gray P, & Zaunders J.J. (2021). The Role of ZEB2 in Human CD8 T Lymphocytes: Clinical and Cellular Immune Profiling in Mowat–Wilson Syndrome. International Journal of Molecular Sciences, 22(10), 5324.

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Multiparametric Stimulation Assay for Cytotoxic T-cell Analysis

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Staining was performed as previously described with slight modifications (12 (link)) on ex vivo blood and mucosal mononuclear cells rested overnight at 37°C, 5% CO₂. For stimulation assays, cells were incubated at 2x10⁶ cells per 200μL R-15 for 5.5 hours in the presence of anti-CD28 (1μg/mL) and anti-CD49d (1μg/mL), anti-CD107a, 1μM GolgiStop^TM (BD Biosciences), brefeldin A (5μg/mL, Sigma Aldrich) and the appropriate antigenic stimulation: Gag-peptide pool (final concentration: 3.5μg/mL of each peptide), or medium containing an equivalent amount of DMSO (peptide solvent) as the negative control. Staphylococcal enterotoxin B (5μg/mL) was used as positive control. Following incubation, cells were stained for surface markers and viability (Aqua Dead cell stain kit, Invitrogen) for 20 minutes at room temperature. Cells were then fixed in 4% paraformaldehyde and permeabilized using FACS Perm 2 (BD Biosciences) prior to intracellular staining for CD3, cytotoxic effectors, chemokines, and cytokines. Staining for cytotoxic effectors following antigenic stimulation (Figs. 3, 4 and Supplemental Figs. 2, 3) utilized staining protocols and antibody clones previously demonstrated to reveal perforin and Granzyme B expression following TCR stimulation (21 (link)). Cells were re-suspended in 1% paraformaldehyde and stored at 4°C in the dark no longer than 24 hours until analysis.

Kiniry B.E., Hunt P.W., Hecht F.M., Somsouk M., Deeks S.G, & Shacklett B.L. (2018). Differential Expression of CD8+ T Cell Cytotoxic Effector Molecules in Blood and Gastrointestinal Mucosa in HIV-1 Infection. Journal of immunology (Baltimore, Md. : 1950), 200(5), 1876-1888.

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Cytokine Profiling in Activated T Cells

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To analyse the cytokine production in T cells, PBMCs were isolated from heparinized venous blood using density-gradient centrifugation over a Ficoll-Hypaque gradient (GE Healthcare). The PBMCs were washed with PBS and resuspended in RPMI 1640 medium with 100 U/mL penicillin, 100 U/mL streptomycin and 10% (v/v) heat-inactivated fetal calf serum (HyClone, Logan, UT, USA). After washing, the cells were stained with PerCP-labeled anti-human CD4 (BD Biosciences) at room temperature for 30 min. After washing with FACS buffer (0.1% [w/v] BSA/PBS), the cells were stimulated with 50 ng/mL phorbol myristate acetate (PMA, Sigma-Aldrich, St. Louis, MO, USA) and 1 µg/mL ionomycin (Sigma-Aldrich) in the presence of BD GolgiStop^TM (BD Biosciences) for 4 h at 37 °C. The stimulated cells were washed with FACS buffer, fixed for 10 min with 4% (v/v) paraformaldehyde, permeabilized with FACS™ Perm 2 (BD Biosciences) according to the manufacturer’s instructions, and stained with FITC-conjugated anti-human IFN–γ, IL-1, IL-6 (BD Biosciences), PE-conjugated anti-human IL-10, IL-17, IFN-γ, and TNF-α (eBioscience) antibodies.

Oh C.S., Kim K., Kang W.S., Woo N.S., Kang P.S., Kim J.S., Kim H.R., Lee S.H, & Kim S.H. (2018). Comparison of the expression of cluster of differentiation (CD)39 and CD73 between propofol- and sevoflurane-based anaesthesia during open heart surgery. Scientific Reports, 8, 10197.

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PBMC Isolation and CD4+ T-cell Characterization

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Peripheral blood mononuclear cells (PBMCs) were isolated using density-gradient centrifugation for 20 min over Biocoll separating solution (Biochrom, Germany). The PBMCs were washed with 1 × PBS (pH 7.4, no calcium, no magnesium), 155.1 mM sodium chloride (NaCl), 2.9 mM sodium phosphate dibasic (Na₂HPO₄-7H₂O), and 1.0 mM potassium phosphate monobasic (KH₂PO₄) solution. After isolating the lymphocytes, the cells were stained with fluorescein isothiocyanate, an anti-human cluster of differentiation 4 antibody (BD Biosciences, USA) at room temperature for 30 min in the dark. After fixation, the cells were permeabilized with FACS™ Perm2 (BD Biosciences, USA) buffer according to the manufacturer’s instructions. After permeabilization, the cells were stained and incubated with the same CHOP antibodies used in the MCF-7 cell line culture, using the same methods.

Oh C.S., Hong S.W., Park S., Kwon Y, & Kim S.H. (2022). Effect of equipotent doses of propofol and sevoflurane on endoplasmic reticulum stress during breast cancer surgery. Korean Journal of Anesthesiology, 75(6), 487-495.

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Multicolor Flow Cytometry Staining

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Cells were stained with LIVE/DEAD fixable Aqua Stain (Life Technologies), followed by surface staining and then fixed and permeabilized with FACS Lyse and FACS Perm II (BD Pharmingen) according to the manufacturer's instructions. Cells were stained with anti-CD3-PE or -APC, anti-CD16-PerCPCy5.5 (clone 3G8; BioLegend), anti-IFNγ-FITC or V450 (clone B27; BD Biosciences), anti-CD56-PEcy7, or anti-CD14-APC or -APC-H7 and fixed using 1% paraformaldehyde. Uncompensated data were collected using a three-laser MACSQuant® Analyser (Miltenyi). Analysis and compensation were performed using FlowJo flow-cytometric analysis software, version 9.9.4 (Tree Star).

Le Gars M., Seiler C., Kay A.W., Bayless N.L., Starosvetsky E., Moore L., Shen-Orr S.S., Aziz N., Khatri P., Dekker C.L., Swan G.E., Davis M.M., Holmes S, & Blish C.A. (2019). Pregnancy-Induced Alterations in NK Cell Phenotype and Function. Frontiers in Immunology, 10, 2469.

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Optimized 12-Color Cytokine Staining Assay

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We have previously published an optimized and validated 12-color intracellular cytokine staining assay to quantify antigen-specific T cells after vaccination(23 (link), 24 (link)). This panel was modified to include anti-IL-17a and anti-IL-22 instead of anti-MIP-1β and anti-CD107a (Supp Table 1). Briefly, cells were first stained with Avid Live/Dead (Life Technologies) viability dye and anti-CD14. After washing, the cells were permeabilized with FACS Perm II (BD) and stained for the remaining markers (CD3, CD4, CD8, CD154, IFN-γ, TNF-α, IL-2, IL-4, IL-17a, and IL-22). Fully stained cells were washed and resuspended in 1% paraformaldehyde. Data were acquired on LSR II (BD) equipped with a high-throughput sampler and configured with violet (405nm, 50mW), blue (488nm, 20mW), green (561nm, 50mW), and red (641nm, 150mW) lasers. Raw data were manually gated in FlowJo (TreeStar Inc.). The raw FCS files and manual gates were imported into the R environment using the OpenCyto framework for data visualization and downstream analysis(25 (link)). For some of the assays with lipid antigens, we noted tandem dye breakdown within APC-Cy7 CD4 leading to spurious signal within the APC IL-4 gate, so this cytokine was not included in downstream analysis. Even though this artifact was not observed with peptide antigens, we removed IL-4 signal from peptide data to maintain consistency.

Seshadri C., Lin L., Scriba T.J., Peterson G., Freidrich D., Frahm N., DeRosa S.C., Moody D.B., Prandi J., Gilleron M., Mahomed H., Jiang W., Finak G., Hanekom W.A., Gottardo R., McElrath M.J, & Hawn T.R. (2015). T-cell responses against mycobacterial lipids and proteins are poorly correlated in South African adolescents. Journal of immunology (Baltimore, Md. : 1950), 195(10), 4595-4603.

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Mass Cytometry Staining Protocol

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Detailed staining protocols have been described (42 (link), 43 (link)). Briefly, cells were transferred to 96-well deep-well plates (Sigma), resuspended in 25 μM cisplatin (Enzo Life Sciences) for 1 min (44 (link)), and quenched with 100% serum. Cells were stained for 30 min on ice, fixed (BD FACS Lyse), permeabilized (BD FACS Perm II), and stained with intracellular antibodies for 45 min on ice. Staining panels are described in table S1. All antibodies were conjugated using MaxPar X8 labeling kits (DVS Sciences). Cells were suspended overnight in iridium interchelator (DVS Sciences) in 2% paraformaldehyde in phosphate-buffered saline (PBS) and washed 1× in PBS and 2× in H₂O immediately before acquisition. Stained cells were analyzed on a mass cytometer (Fluidigm).

Strauss-Albee D.M., Fukuyama J., Liang E.C., Yao Y., Jarrell J.A., Drake A.L., Kinuthia J., Montgomery R.R., John-Stewart G., Holmes S, & Blish C.A. (2015). Human NK cell repertoire diversity reflects immune experience and correlates with viral susceptibility. Science translational medicine, 7(297), 297ra115.

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Phenotypic Analysis of Cryopreserved PBMCs

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Cryopreserved PBMCs from non-pregnant and pregnant women in discovery and validation cohort were thawed and cells were transferred to 96-well deep-well-plates, resuspended in 25 μM cisplatin (Enzo Life Sciences) for 1 min and quenched with 100% serum. Cells were stained for 30 min, fixed (BD FACS Lyse), permeabilized (BD FACS Perm II), and stained with intracellular antibodies for 45 min on ice. Staining panels are described in Tables S3, S4. All antibodies were conjugated using MaxPar X8 labeling kits (DVS Sciences). Cells were suspended overnight in iridium intercalator (DVS Sciences) in 2% paraformaldehyde in phosphate-buffered saline (PBS) and washed 1 × in PBS and 2 × in H₂O immediately before acquisition on a CyTOF-1 (Fluidigm).

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