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Permeabilization buffer 3

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Permeabilization Buffer III is a buffer solution designed for permeabilizing cells in preparation for intracellular staining or analysis. It is a ready-to-use formulation that aids in the temporary disruption of cell membranes, allowing for the introduction of antibodies or other reagents into the cell interior.

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Lab products found in correlation

Cytofix fixation buffer, by BD (3 mentions) Cytofix buffer, by BD (2 mentions) Fetal bovine serum (fbs), by Wisent (1 mentions) Human serum, by Atlanta Biologicals (1 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions) Rpmi 1640, by Thermo Fisher Scientific (1 mentions) Lsr 2, by BD (1 mentions) Anti human cd45 apc, by BD (1 mentions) Lsrii flow cytometer, by BD (1 mentions) N7 548, by BD (1 mentions) Stain buffer, by BD (1 mentions) Cytofix, by BD (1 mentions) Perk1 2 pt202 py204, by BD (1 mentions) Fixation buffer, by BD (1 mentions) Paraformaldehyde pfa, by Merck Group (1 mentions)

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Permeabilization buffer (110 citations) Buffer III (2 citations)

7 protocols using permeabilization buffer 3

1

Quantifying NF-kB Activation in CD4 T Cells

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PBMCs were rested overnight in complete medium (RPMI 1640, Life Technologies) supplemented with 10% Fetal Bovine Serum (Wisent) and 1% Penicillin/Streptomycin (Life Technologies) and exposed to 0.27nM-350nM PMA, 0.69nM-900nM bryostatin, 0.69nM-900nM ingenol or 0.069μM-90μM prostratin for 15min at 37°C in PBS/2% Human Serum (Atlanta Biologicals, 540110). Antibody to CCR7 and LIVE/DEAD reagent were added to the culture medium during the stimulation. Cells were then fixed (10min, 37°C) with the Cytofix Fixation Buffer (BD Biosciences, 554655) and stained (1h, room temperature) with antibodies to cell surface markers (CD3, CD4, CD8 and CD27). Cells were permeabilized (20min, on ice) with the ice-cold Permeabilization Buffer III (BD Biosciences, 558050), and rehydrated (15min, on ice) in PBS/Human Serum 10%. Finally, PBMCs were stained (30min, RT) with anti-pS129 NF-κB and anti-CD45RA antibodies. Levels of pNF-κB in gated subsets of CD4 T cells were determined by flow cytometry on a BD LSR II.
Pardons M., Fromentin R., Pagliuzza A., Routy J.P, & Chomont N. (2019). Latency Reversing Agents Induce Differential Responses in Distinct Memory CD4 T Cell Subsets in Individuals on Antiretroviral Therapy. Cell reports, 29(9), 2783-2795.e5.
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2

Multiparametric Flow Cytometry Analysis

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Cells isolated from humanized mice were prepared for flow cytometry as described above and stained with the following extracellular markers: anti-mouse CD45-BV605 (BD), anti-human CD45-APC (BD), anti-human CD3-APC-eFluor780 (Thermo Fisher), anti-human CD8-AF700 (BD), and anti-human CD33-BV711 (BD). Cells were fixed with Cytofix buffer (BD) at 37°C for 10 min. Following this, cells were permeabilized with permeabilization buffer III (BD) for 30 min at 4°C and stained with the following phosflow antibodies: anti-human pSTAT1-BV421 (BD), anti-human pp65-PerCP-eFluor710 (Life Technologies), anti-human pIRF3 S396-PE (Cell Signaling, MA, USA), and anti-human pTBK-1 (Cell Signaling).
Skelton J.K., Ortega-Prieto A.M., Kaye S., Jimenez-Guardeño J.M., Turner J., Malim M.H., Towers G.J, & Dorner M. (2019). Kinetics of Early Innate Immune Activation during HIV-1 Infection of Humanized Mice. Journal of Virology, 93(11), e02123-18.
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3

Measuring NF-κB Phosphorylation in T Cells

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Blood mononuclear cells were rested for 3 hours (RM cells) or 14 hours (human cells) and exposed to increasing doses of GSK445A for 20 minutes (37°C). LIVE/DEAD staining was performed at the same time than the stimulation. Cells were then fixed with Cytofix Fixation Buffer (BD Biosciences, 554655) (10 minutes, 37°C) and stained with anti-CD3 SP34.2 A700, CD4 L200 BUV395, CD8 RPA-T8 PB for RM cells, and with anti-CD3 UCHT1 A700, CD4 S3.5 Qdot605, CD8 RPA-T8 PB for human cells (45 minutes, RT). Cells were permeabilized for 20 minutes on ice with ice-cold Permeabilization Buffer III (BD Biosciences, 558050), and rehydrated in PBS/Human Serum 10% (Atlanta Biologicals, 540110) for 15 minutes on ice. Finally, PBMCs were stained (30 minutes, RT) with anti-pS529 NF-κB antibody. Data were collected on an LSR-II flow cytometer (BD Biosciences) and analysis performed using FlowJo software (Tree Star).
Okoye A.A., Fromentin R., Takata H., Brehm J.H., Fukazawa Y., Randall B., Pardons M., Tai V., Tang J., Smedley J., Axthelm M., Lifson J.D., Picker L.J., Favre D., Trautmann L, & Chomont N. (2022). The ingenol-based protein kinase C agonist GSK445A is a potent inducer of HIV and SIV RNA transcription. PLoS Pathogens, 18(1), e1010245.
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4

Multiparametric Flow Cytometry of Phosphoproteins

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Samples were fixed with Cytofix (554655, BD) for 12 min at 37°C, spun, decanted, resuspended in Permeabilization Buffer III (558050, BD), and kept at 4°C for 30 min or at −80°C for longer periods. They were then washed thrice in Stain Buffer (554656, BD) and split into two panels before simultaneous labeling for surface, Foxp3, and phosphorylated signaling molecules.
Phospho antibody panels were (i) pERK1/2-PE (BD, 20A) and pAkt S473-AlexaFluor 647 (BD, M89-61) or (ii) pS6-PE (N7-548, BD) and pPDK1-AlexaFluor 647 (J66-653.44.17, BD). Phospho antibody test volumes were 4 μL per 2.5 × 105 cells.
Zwang N.A., Zhang R., Germana S., Fan M.Y., Hastings W.D., Cao A, & Turka L.A. (2016). Selective Sparing of Human Tregs by Pharmacologic Inhibitors of the Phosphatidylinositol 3-Kinase and MEK Pathways. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 16(9), 2624-2638.
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5

TIGIT Modulation of NK-MDSC Interactions

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NK cells from healthy blood donors were co-cultured with MDSCs or monocytes at a 2:1 ratio in the presence of IL-15 (10 ng/ml) and in the presence or absence of catalase (200 IU/ml) or blocking antibodies against TIGIT (10 ug/ml) for 5 days. Cells were then washed, rested for 4 hours, and stimulated with anti-CD16 agonist antibody for 10 and 30 min. before analysis of Zap-70 and ERK1/2 phosphorylation respectively. Cells were fixed and permeabilized with BD fixation buffer and permeabilization buffer III and stained for pZap-70 (pY319)/Syk (pY352) and pERK1/2 (pT202/pY204) according to the manufacturer’s instructions (BD biosciences).
Sarhan D., Cichocki F., Zhang B., Yingst A., Spellman S.R., Cooley S., Verneris M.R., Blazar B.R, & Miller J.S. (2016). Adaptive NK cells with low TIGIT expression are inherently resistant to myeloid-derived suppressor cells. Cancer research, 76(19), 5696-5706.
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6

Dissociation and Phospho-ERK Profiling of HGSOC Tumors

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HGSOC tumor specimen (n=10–22) were dissociated and aliquoted in FACS V tubes with R-10 supplemented with αPD-1 antibody, in the presence or absence of p2TA peptide (as described above). After a quick spin, tubes were incubated in thermal bath at 37°C for 30, 45 and 60 minutes. At each time point, cells were immediately fixed with paraformaldehyde (PFA, Sigma-Aldrich) for 15 minutes at RT, then washed and permeabilized with 1 mL Permeabilization buffer III (BD Biosciences), for 30 minutes on ice. After 2 washes with FACS buffer and Fc blocking, cells were stained with phosphor-ERK-specific antibodies (20A, BD Biosciences) for 1h at RT. After washing, cells were stained for phenotype markers CD45, CD3, CD8, CD28 (antibodies as reported above), and analyzed by FACS (Gallios, Beckman Coulter).
Duraiswamy J., Turrini R., Minasyan A., Barras D., Crespo I., Grimm A.J., Casado J., Genolet R., Benedetti F., Wicky A., Ioannidou K., Castro W., Neal C., Moriot A., Renaud-Tissot S., Anstett V., Fahr N., Tanyi J.L., Eiva M.A., Jacobson C.A., Montone K.T., Wulff Westergaard M.C., Svane I.M., Kandalaft L.E., Delorenzi M., Sorger P.K., Färkkilä A., Michielin O., Zoete V., Carmona S.J., Foukas P.G., Powell DJ J.r., Rusakiewicz S., Doucey M.A., Laniti D.D, & Coukos G. (2021). Myeloid Antigen-Presenting Cell Niches Sustain Antitumor T Cells and License PD-1 Blockade via CD28 Costimulation. Cancer cell, 39(12), 1623-1642.e20.
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7

Intracellular Protein Staining Protocol

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For granzyme B, cells were first stained for surface proteins, fixed in Cytofix buffer (BD Biosciences), washed and incubated in saponin buffer (staining buffer +0·1% saponin), and stained in saponin buffer plus anti‐granzyme B antibody (or a matched isotype control). Cells were resuspended in 0·5% paraformaldehyde (in staining buffer) prior to analysis. For other intracellular proteins, cells were stained for surface proteins if required and then fixed in Cytofix fixation buffer (BD Biosciences), according to the manufacturer's instructions. For time‐course experiments, fixed cells were stored at 4°C and all cells within an experiment were stained at the same time. Samples were then resuspended in permeabilization buffer III (BD Biosciences) and permeabilized on ice for 30 min, followed by staining, washing and analysis.
Wantoch M., Wilson E.B., Droop A.P., Phillips S.L., Coffey M., El‐Sherbiny Y.M., Holmes T.D., Melcher A.A., Wetherill L.F, & Cook G.P. (2022). Oncolytic virus treatment differentially affects the CD56dim and CD56bright NK cell subsets in vivo and regulates a spectrum of human NK cell activity. Immunology, 166(1), 104-120.
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