Cytofix cytoperm protocol

Manufactured by BD

The Cytofix/Cytoperm™ protocol is a laboratory technique used to fix and permeabilize cells, allowing for intracellular staining and analysis. It provides a standardized method for preparing cells for flow cytometry or other analytical procedures that require access to intracellular targets.

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6 protocols using cytofix cytoperm protocol

NK Cell Activation Assay Protocol

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Ninety-six-well Maxisorp® plates were coated with anti-CD16 monoclonal antibodies (mAbs) (clone 3G8, BioLegend), anti-NKp30 mAbs (clone 210847, R&D), or the respective isotype control mAbs (each at 2.5 μg/mL in 50 μL of sterile PBS 1X). Plates were incubated overnight at 4°C. Thawed PBMC were incubated overnight with Cellgro medium (Cellgenix) at 2 × 10⁶ cells/mL. The next day, cells were incubated in the antibody-coated plates for 5 h in the presence of anti-CD107a, IL-2 (1,000 iu/mL), and Brefeldin A. Cells were then stained with anti-CD3 (BW264/56), anti-CD56 (N901), anti-CD45 (J33) and LIVE/DEAD® fixable yellow dead cell dye (Molecular Probes, Life Technologies). For intracellular staining, cells were fixed and permeabilized according to the Cytofix/Cytoperm™ protocol (BD Bioscience) and then stained with anti-IFN-γ (4SB3) and anti-TNF-α (Mab11) fluorochrome-conjugated antibodies.

Besse B., Charrier M., Lapierre V., Dansin E., Lantz O., Planchard D., Le Chevalier T., Livartoski A., Barlesi F., Laplanche A., Ploix S., Vimond N., Peguillet I., Théry C., Lacroix L., Zoernig I., Dhodapkar K., Dhodapkar M., Viaud S., Soria J.C., Reiners K.S., Pogge von Strandmann E., Vély F., Rusakiewicz S., Eggermont A., Pitt J.M., Zitvogel L, & Chaput N. (2015). Dendritic cell-derived exosomes as maintenance immunotherapy after first line chemotherapy in NSCLC. Oncoimmunology, 5(4), e1071008.

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Comprehensive Immunophenotyping of Bone Marrow Cells

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BM-MNC (1.5×10⁶ cells/sample) were stained with appropriate conjugated antibodies in PBS with 2% BSA. Specifically, human BM-MNC were stained with: anti-CD33-PE-Cy7, anti-CD34-PerCP-Cy5.5, anti-CD14-BV510, anti-CD71-BV421, anti-CD38-BV711, anti-CD235a-BUV395, anti-PD-1-FITC, and anti-PD-L1-APC (BD Biosciences). Gating strategies for the identification of myeloid cells, HSPCs, and erythroid progenitors are shown in Supplementary Figure S1. Mouse BM cells were stained with: anti-c-Kit-PerCP-Cy5.5, anti-Sca-1-PE, Lin-APC (including anti-CD3ε, anti-CD11b, anti-CD45R/B220, anti-TER-119, anti-Gr-1), anti-PD-1-BV421, anti-PD-L1BV711, anti-PD-L2-BUV395, and anti-CD16/32-PE-Cy7 (BD Biosciences). Cell viability was determined using near infrared live/dead dye (BD Biosciences) and the negative (live cell) population was used for further analysis. Samples were acquired on an LSR II flow cytometer and analyzed using FlowJo 9.9.3 software. Intracellular staining with PE-conjugated anti-active caspase 3 was performed using Cytofix/Cytoperm™ protocol following initial cell surface receptor staining (BD Biosciences). For PD-1/PD-L1 ligation experiments, 2 million BM-MNC were plated per well in 24-well plates coated with recombinant human PD-L1 (2 μg/mL) for 24 hr at 4 °C.

Cheng P., Eksioglu E.A., Chen X., Kandell W., Le Trinh T., Cen L., Qi J., Sallman D.A., Zhang Y., Tu N., Adams W.A., Zhang C., Liu J., Cleveland J.L., List A.F, & Wei S. (2019). S100A9-induced overexpression of PD-1/PD-L1 contributes to ineffective hematopoiesis in myelodysplastic syndromes. Leukemia, 33(8), 2034-2046.

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Immunophenotypic Analysis of Bone Marrow Cells

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BM-MNC (1.5 × 10⁶ cells/sample) were stained with appropriate conjugated antibodies in PBS with 2% BSA. Specifically, human BM-MNC were stained with anti-CD33-PE-Cy7, anti-CD34-PerCP-Cy5.5, anti-CD14-BV510, anti-CD71-BV421, anti-CD38-BV711, anti-CD235a-BUV395, anti-PD-1-FITC, and anti-PD-L1-APC (BD Biosciences). Gating strategies for the identification of myeloid cells, HSPCs, and erythroid progenitors are shown in Supplementary Figure S1. Mouse BM cells were stained with anti-c-Kit-PerCP-Cy5.5, anti-Sca-1-PE, Lin-APC (including anti-CD3ε, anti-CD11b, anti-CD45R/B220, anti-TER-119, anti-Gr-1), anti-PD-1-BV421, anti-PD-L1-BV711, anti-PD-L2-BUV395, and anti-CD16/32-PE-Cy7 (BD Biosciences). Cell viability was determined using near-infrared live/dead dye (BD Biosciences) and the negative (live cell) population was used for further analysis. Samples were acquired on an LSR II flow cytometer and analyzed using FlowJo 9.9.3 software. Intracellular staining with PE-conjugated anti-active caspase-3 was performed using the Cytofix/Cytoperm™ protocol following initial cell surface receptor staining (BD Biosciences). For PD-1/PD-L1 ligation experiments, 2 million BM-MNC were plated per well in 24-well plates coated with recombinant human PD-L1 (2 μg/mL) for 24 h at 4 °C.

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Comprehensive Cellular Immuno-Phenotyping Protocol

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Cellular immuno-phenotyping and staining for BrdU incorporation were performed as described [5 (link), 23 (link)]. Briefly, 200 μl of EDTA–anticoagulated whole blood were washed with PBS (Catalogue number 21–040-CV, Corning, Corning, NY) containing 2% FBS (Catalogue number 16000044, Thermo Fisher Scientific, Vacaville, CA) and incubated with surface monoclonal antibodies (Supplemental Table 1) at room temperature for 20 minutes. RBCs were lysed with FACS lysing solution (10× stock, Catalogue Number 349202, BD Biosciences, San Jose, CA) prepared to 1× final concentration in deionized water, and the remaining cells were permeabilized using a three-step Cytofix/Cytoperm protocol per manufacturer instructions (BD Biosciences). For analysis of BrdU incorporation, cells were incubated with DNase I (Catalogue number DN25, Sigma-Aldrich) at 37°C for one hour and then stained with anti-BrdU antibody for 20 min at room temperature (Supplemental Table 1). After washing, cells were fixed in 250 μl of 1% paraformaldehyde in PBS. Samples were acquired with a LSRFortessa flow cytometer (BD Biosciences) and data were analyzed with FlowJo software version 10 (FlowJo, LLC, Ashland, OR).

He Z., Fahlberg M.D., Takahashi N., Slisarenko N., Rout N., Didier E.S, & Kuroda M.J. (2021). Declining neutrophil production despite increasing G-CSF levels is associated with chronic inflammation in elderly rhesus macaques. Journal of leukocyte biology, 109(6), 1033-1043.

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Multiparameter Immune Cell Profiling

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Single-cell suspensions (1 × 10⁶ cells) was incubated at 4°C for 15 min with α-CD16/32 (clone 2.4G2, from Bioexpress) to reduce non-specific binding. Cells were labeled with various combinations of directly conjugated monoclonal antibodies and incubated at 4°C for 25 min. The following monoclonal antibodies were used: α-NK1.1 (PK136), α-CD3 (145-2C11), α-TCRβ (H57-597), α-CD8a (53–6.7), α-CD49b (DX5), α-CD27 (LG.7F9), α-CD11b (M1/70), α-Ly49D (4E5), α-Ly49G2 (4D11), α-Ly49H (3D10), α-NKG2A-B6 (16a11), α-NKG2D (CX5), α-IL-10 (JES5-16E3), α-IFNγ (XMG1.2), and α-CD107a (1D4B) from eBioscience, α-CD19 (1D3), α-Ly49C/I (5E6), α-CD4 (RM4-5), and α-CD49a (Ha31/8) from BD Biosciences, Fixable Yellow Live/Dead from Invitrogen. For intracellular IL-10 and IFNγ measurements in vivo, leukocytes were harvested and incubated in RP-10 medium (RPMI-1640, 10% FBS, 1X penicillin/streptomycin, 1% L-Glutamine, 10 mM HEPES, 50 μM β-mercaptoethanol) containing 5 μg/ml brefeldin A for 4 h, followed by staining for intracellular IL-10 or IFNγ using BD Cytofix/Cytoperm protocol. The stained cells were acquired using LSRFortessa or FACSCelesta (BD Biosciences) and analyzed using Kaluza software v3 (Beckman Coulter) or FlowJo (Tree Star).

Ali A.K., Komal A.K., Almutairi S.M, & Lee S.H. (2019). Natural Killer Cell-Derived IL-10 Prevents Liver Damage During Sustained Murine Cytomegalovirus Infection. Frontiers in Immunology, 10, 2688.

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Murine Bone Marrow Cell Stimulation

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Cell suspensions from mouse BM were washed and suspended in 2% FCS RPMI. Cells were warmed at 37°C for 5min and incubated at 37°C (10⁶ cells/well in 96w U bottom plates) with the indicated doses of anti-IgM F(ab)₂ (Jackson Immunoresearch), sHEL (Sigma) for 5 minutes, BpV(phen) (Sigma) for 30 minutes. Subsequent surface and intracellular staining were performed using the BD Cytofix/Cytoperm protocol.

Anzilotti C., Swan D.J., Boisson B., Deobagkar-Lele M., Oliveira C., Chabosseau P., Engelhardt K.R., Xu X., Chen R., Alvarez L., Berlinguer-Palmini R., Bull K.R., Cawthorne E., Cribbs A.P., Crockford T.L., Dang T.S., Fearn A., Fenech E.J., de Jong S.J., Lagerholm B.C., Ma C.S., Sims D., van den Berg B., Xu Y., Cant A.J., Kleiner G., Leahy T.R., de la Morena M.T., Puck J.M., Shapiro R.S., van der Burg M., Chapman J.R., Christianson J.C., Davies B., McGrath J.A., Przyborski S., Koref M.S., Tangye S.G., Werner A., Rutter G.A., Padilla-Parra S., Casanova J.L., Cornall R.J., Conley M.E, & Hambleton S. (2019). An essential role for the Zn2+ transporter ZIP7 in B cell development. Nature immunology, 20(3), 350-361.

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