Cells were incubated with αCD16/32 (clone 2.4G2; hybridoma supernatant) and plated on U-bottom 96-well plates at ≤ 3 × 106 cells/well in complete RPMI. Where indicated, cells were stained with Kb-SIINFEKL tetramer APC (NIH tetramer core) at 37°C for 30 min in the presence of αCD8α (53–6.7; Biolegend). For live-dead and surface staining, cells were washed with media, and stained in media for 20 min at RT with Fixable Viability Dye 780 (eBioscience) and surface antibodies for CD19 (6D5, Biolegend), CD8α (53–6.7, Biolegend), CD44 (IM-7; Tonbo), CD127 (A7R34, Tonbo), KLRG1 (2F1/KLRG1, Biolegend), CD45.1 (A20, Biolegend), CD45.2 (104, Biolegend), CD122 (TM-b1, Biolegend). After wash with RPMI, cells were fixed and permeabilized for 45 min at RT in Foxp3/Transcription Factor 1× Fix/Perm solution (Tonbo), followed by wash with 1× Flow Cytometry Perm Buffer (Tonbo) and intracellular staining for 45 min at RT with intracellular antibodies, including TCF1 (C63D9; Cell Signaling Technology), FOXO1 (C29H4, Cell Signaling Technology), T-bet (4B10, Biolegend), EOMES (Dan11mag; eBioscience). The cells were washed twice and resuspended in 1× Flow Cytometry Perm Buffer (Tonbo). Flow cytometry data was acquired on a four-laser (405, 488, 561, 638 nm) CytoFlex S (Beckman Coulter) and analyzed using FlowJo software (BD Biosciences).
Flow cytometry perm buffer
Manufactured by Cytek Biosciences
Sourced in United States
The Flow Cytometry Perm Buffer is a reagent used in flow cytometry applications to permeabilize cells, allowing for the intracellular staining and analysis of cellular proteins and other intracellular targets. The buffer works by creating temporary pores in the cell membrane, facilitating the access of fluorescently labeled antibodies or other probes to the cells' interior.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by BioLegend (2 mentions)
A1 laser scanning confocal microscope, by Nikon (2 mentions)
Bv421 anti flag, by BioLegend (2 mentions)
Facsverse flow cytometer, by BD (2 mentions)
Alexa fluor 647 conjugated anti active caspase 3 mab c92 605, by BD (2 mentions)
Fixable viability dye 780, by Thermo Fisher Scientific (1 mentions)
Cd19 6d5, by BioLegend (1 mentions)
Eomes dan11mag, by Thermo Fisher Scientific (1 mentions)
Cytoflex s, by Beckman Coulter (1 mentions)
α cd8α, by BioLegend (1 mentions)
Cd44 im7, by Cytek Biosciences (1 mentions)
Cd127 a7r34, by Cytek Biosciences (1 mentions)
Klrg1 2f1 klrg1, by BioLegend (1 mentions)
Cd45.1 a20, by BioLegend (1 mentions)
Cd45.2 104, by BioLegend (1 mentions)
T bet, by BioLegend (1 mentions)
Foxo1, by Cell Signaling Technology (1 mentions)
Collagenase type 4, by Worthington (1 mentions)
Anti mouse cd16 32 antibody, by BioLegend (1 mentions)
Macsquant analyzer, by Miltenyi Biotec (1 mentions)
7 protocols using flow cytometry perm buffer
1
Characterization of CD8+ T Cell Subsets
2
Multiparameter Flow Cytometry of Lymphocytes
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Spleens were digested with 1 mg/ml Collagenase type IV (Worthington) and 40 U/ml DNase I (Roche Diagnostics) in HBSS (−) at 37°C for 30 min. Single-cell suspension were incubated with anti-mouse CD16/32 antibody (BioLegend) to prevent nonspecific binding of antibodies and stained with fluorescence-conjugated antibodies in 0.5% BSA, 5 mM EDTA in PBS. The following conjugated antibodies were used for the staining: B220 (RA3-6B2), CD23 (B3B4), CD21 (7E9), CD138 (281-2), CD95 (SA367H8), GL7 (GL7), CD3 (145-2C11), CD4 (GK1.5), CD8 (53-6.7), CD11c (N418), MHCII (M5/144.15.2), PDCA (9.27E+02), CD11b (M1/70), Ly6G (1A8), CD44 (IM7), CD62L (MEL-14), PD-1 (29F.1A12), CD25 (3C7) (BioLegend), Peanut Agglutinin (PNA, Vector Laboratories), and DAPI (BioLegend). For intracellular staining, cells were incubated with Transcription Factor Fix/Perm Working Buffer (TONBO Biosciences) at room temperature for 30 min. After washing twice with Flow Cytometry Perm Buffer (TONBO Biosciences), cells were stained with APC-conjugated anti-Blimp1 (5E7, BioLegend) or PE-conjugated anti-Foxp3 (3G3, TONBO Biosciences) at room temperature for 20 min in the dark. After washing, cells were analyzed on MACSQuant Analyzer (Miltenyi Biotec) and FlowJo software (BD Bioscience). An example of the gating strategy for B cells, T cells and myeloid cells is shown in Figures S2 S5
3
FLAG-tagged RARα Localization in Th17 Cells
4
Intracellular HIV-1 p24 and Caspase-3 Assay
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Intracellular HIV-1 p24 levels and active form of caspase-3 expression were determined as previously described (Hattori et al., 2009 (link); Duro et al., 2010 (link); Matsuda et al., 2015 (link)). In brief, ACH-2 cells or U1 cells (2.5 × 105 cells/mL) were fixed with 1% paraformaldehyde/phosphate buffered saline (PBS) for 20 min and were permeabilized with Flow Cytometry Perm Buffer (TONBO biosciences, San Diego, CA, United States). After incubation on ice for 5 min, the cells were stained with anti-HIV-1 p24 (24-4)-FITC mAb (Santa Cruz Biotechnology, Dallas, TX, United States) and/or with Alexa Fluor 647-conjugated anti-active caspase-3 mAb (C92-605) (BD Pharmingen, San Diego, CA, United States) for 30 min on ice. Cells were then analyzed on a BD FACSVerse flow cytometer (BD Biosciences, Franklin Lakes, NJ, United States). The corrected data were analyzed with FlowJo software (Tree Star, San Carlos, CA, United States).
5
Multiparametric Flow Cytometry Analysis of HIV-1 Infection
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The ratios of intracellular HIV-1 p24+ cells, GFP+ cells, and the active form of caspase-3 expression were determined as previously described (Hattori et al., 2018 (link); Matsuda et al., 2019 (link)). Briefly, Jurkat/NL or JLTRG/NL cells were washed twice with phosphate buffered salts (PBS) and stained with Ghost Dye Red 780 (TONBO Biosciences, San Diego, CA) for 30 min at 4°C. The cells were then fixed with 1% paraformaldehyde/PBS for 20 min, and permeabilized in a flow cytometry perm buffer (TONBO Biosciences). After 5-min incubation at room temperature (25–30°C), cells were stained with anti-HIV-1 p24 (24-4)-fluorescein isothiocyanate (FITC) monoclonal antibody (mAb; Santa Cruz Biotechnology) and/or Alexafluor 647-conjugated anti-active caspase-3 mAb (C92-605; BD Pharmingen, San Diego, CA) for 30 min on ice. For propidium iodide (PI)/annexin V staining, cells were washed twice with PBS and resuspended in annexin V binding buffer (BioLegend) at a concentration of 1 × 107 cells/mL. The cells were then stained with FITC annexin V (BioLegend) and PI solution (BioLegend) for 15 min at room temperature. Cells were analyzed using a BD FACSVerse flow cytometer (BD Biosciences, Franklin Lakes, NJ). Data collected were analyzed using FlowJo software (Tree Star, Inc., Ashland, OR).
6
FLAG-tagged RARα Localization in Th17 Cells
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WT or ligand-binding defective (ΔLB, G303E) forms of human RARα 45 (link) with a N-terminal FLAG-tag were cloned into pMSCV-Thy1.1 retroviral vectors. Retroviral supernatants containing viral particles were produced from plasmid-transfected Platinum E cells. Naïve T helper cells were cultured for 16 h in cRPMI with plate-bound anti-CD3 (5 μg/mL) and soluble anti-CD28 (2 μg/mL), then transduced with retroviral supernatants in the presence of polybrene (8 μg/mL) by spin transduction (90 m, 2300 rpm, 32oC). Cells were rested for 1–2 h after transduction, then reactivated in a Th17-polarizing condition with or without 10 nM RA for 20 h. Cells were then harvested and stained for surface Thy1.1 (clone OX-7), fixed with 1% paraformaldehyde for 1 hour, permeabilized with flow cytometry perm buffer (Tonbo Biosciences; San Diego, CA) and stained with BV421 anti-FLAG (BioLegend, clone L5) and DRAQ5. Cells were spun onto Cell-Tak coated glass slides and imaged with a Nikon A1 laser scanning confocal microscope with 60× objective. Cellular localization of FLAG-RARα was analyzed using ImageJ.
7
Cell Cycle Analysis by Flow Cytometry
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FoxP3/Transcription Factor Staining Buffer Kit (Tonbo Biosciences; cat. no. TNB-0607) is a paraformaldehyde/saponin based fixation/permeabilization buffer set for intracellular staining, and was adapted here for cell cycle analysis. Briefly, cells stained with extracellular antibodies were fixed with 1X Tonbo Foxp3/Transcription Factor Fix/Perm buffer for 45 min at 4°C, permeabilized/stained with PE anti-ki-67 antibody (Biolegend; cat. no. 652403) diluted in 1X Tonbo Flow Cytometry Perm Buffer for 45 min in the dark at room temperature. Cells were then washed and stained with 1 μM DAPI (Biolegend; cat. no. 422801) for 10 min prior to flow cytometric analysis. Ki-67 is a nuclear protein associated with cellular proliferation, and is expressed on cells that have entered the cell cycle, but not on quiescent G0 cells (Kim and Sederstrom, 2015 (link)). DAPI is a nuclear dye that can distinguish cells that have undergone DNA replication. We recommend avoiding separation of e10.5 tissues into fewer than 4 ee as the fix/perm process results in loss of cells.
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