Gill leucocytes were isolated as described above. Cells were fixed with fixation buffer (Biolegend) and washed twice with permeabilization wash buffer (Biolegend) following the manufacturer's instructions. Permeabilized cells were stained with monoclonal mouse anti-trout IgT (1 μg ml−1), or mouse IgG2b as isotype control (Biolegend; 1 μg ml−1) in permeabilization wash buffer at 4 °C for 30 min. Secondary antibody APC-goat anti-mouse IgG2b (Jackson ImmunoResearch Laboratories) was added after washing and incubated for 30 min at 4 °C. Analyses of stained leucocytes were performed by flow cytometry using a FACSCanto II and FlowJo software.
Permeabilization wash buffer
Manufactured by BioLegend
Sourced in United States, United Kingdom
Permeabilization Wash Buffer is a solution designed to facilitate the permeabilization of cells during immunostaining or flow cytometry procedures. It is used to create pores in cell membranes, allowing antibodies or other reagents to access the intracellular compartments of the cells.
Automatically generated - may contain errors
Lab products found in correlation
Fixation buffer, by BioLegend (30 mentions)
Cell staining buffer, by BioLegend (4 mentions)
Brefeldin a, by BioLegend (3 mentions)
Fixable viability dye efluor 780, by Thermo Fisher Scientific (3 mentions)
Golgiplug, by BD (3 mentions)
Allophycocyanin apc conjugated anti mouse ifn γ antibody, by BioLegend (3 mentions)
Phycoerythrin pe conjugated anti mouse il 4 antibody, by BioLegend (3 mentions)
Tryple, by Thermo Fisher Scientific (2 mentions)
Lsrfortessa, by BD (2 mentions)
Facscanto 2 flow cytometer, by BD (2 mentions)
Facsdiva v6, by BD (2 mentions)
Golgistop, by BD (2 mentions)
Permeabilization wash buffer, by BD (2 mentions)
Facsdiva software, by BD (2 mentions)
Monensin, by BioLegend (2 mentions)
Cell activation cocktail with brefeldin a, by BioLegend (2 mentions)
Facsaria flow cytometer, by BD (2 mentions)
Phorbol 12 myristate 13 acetate pma, by Merck Group (2 mentions)
Ionomycin, by Merck Group (2 mentions)
True nuclear transcription factor buffer set, by BioLegend (2 mentions)
66 protocols using permeabilization wash buffer
1
Isolation and Staining of Gill Leucocytes
2
Listeria-Specific T Cell Responses
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Spleens were removed on the indicated days post Lm-OVA infection and disrupted into single-cell suspensions. Spleen cells were treated with Tris-ammonium chloride to lyse red blood cells. Total Listeria-specific T cells were determined by intracellular cytokine staining for IFN-γ after 5 hours of stimulation at 37°C with 200 nM OVA257-264 or 5 mM LLO190-201 in the presence of brefeldin A (Biolegend, San Diego, CA, USA). For MHC I tetramer staining, cells were incubated 45 min at room temperature with MHC class I tetramers loaded with OVA257-264 (generously provided by Dr. John Harty, University of Iowa, Iowa City, IA) followed by anti-CD8 and anti-Thy1.2 antibodies for 15 min at room temperature and then fixed with Fixation Buffer (Biolegend, San Diego, CA, USA). For phenotypic analysis, after incubation with peptides cells were surface stained with the indicated antibodies then fixed and permeabilized with Fixation Buffer. Intracellular staining for IFN-γ, IL-2, and TNF were done in the presence of 1X Permeabilization Wash Buffer (Biolegend, San Diego, CA, USA). Cell surface expression of CD62L was detected by incubating cells with 0.1 mM TAPI-2 (Peptides International, Louisville, KY, USA) for 30 min prior to and during stimulation with peptides.
3
Flow Cytometric Analysis of iPSC-Derived Cardiomyocytes
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SMA and Control patient iPSC-derived cardiomyocytes were dissociated using TrypLE (Gibco, cat 12605-010). Disaggregated cells were centrifuged and resuspended in fixation buffer (Biolegend, cat 554655) for 15 min at room temp, washed 3 times in 1X permeabilization wash buffer (Biolegend, cat 421002, and resuspended in permeabilization wash buffer containing FITC conjugated anti- Cardiac Troponin (1:20, Miltenyi Biotec, cat 130-106-689) for 1 h at room temp protected from light. Cells were read in cell staining buffer (Biolegend, cat 420201) on BD Accuri C6 Flow Cytometer CFlow Plus software. Unstained cells were used as a negative control.
4
Multiparametric Flow Cytometry of Immune Cells
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Single-cell suspensions of skin and dLN cells were incubated with anti-CD16/CD32 (TONBO Biosciences) for 5 min, and then stained using the fluorochrome-conjugated antibodies described in Table S1 . Intracellular GzmB and CTLA-4 were stained using Fixation Buffer and 10 × Permeabilization Wash Buffer (BioLegend) as described in the manufacturer's protocol. The fluorescence intensity of KikGR-Red was reduced by fixation, thus, the proportion of KikGR-Red+ cells was lower in samples subjected to intracellular staining than in fresh samples. Dead cells were stained with 7-AAD (eBioscience) or propidium iodide (Wako) and excluded from our analyses. Stained samples were analyzed using a SP6800 Spectral Cell Analyzer (SONY). All data were exported as FCS files and analyzed using FlowJo software (Tree Star).
5
Regulatory T Cell Analysis by Flow Cytometry
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Antibody-stained samples (see Supplementary Methods ) were analyzed using a LSRFortessa (BD Biosciences) or SP6800 (SONY) flow cytometer. Data were analyzed using FlowJo software (Tree Star). Tregs were identified as CD4+ hCD2+ cells falling within a lymphocyte forward scatter and side scatter gate.
To detect IL-10-producing cells, cells from the dLN were incubated at 37° C for 6 h with DNBS (50 μg/mL, Alfa Aesar) or PMA and ionomycin (50 ng/mL and 1 μg/mL), with the addition of brefeldin A (5 μg/mL, BioLegend) for the last 4 h. Intracellular staining for GzmB and IL-10 was performed using Fixation Buffer and 10× Permeabilization Wash Buffer (BioLegend), as described in the manufacturer’s protocol.
To detect IL-10-producing cells, cells from the dLN were incubated at 37° C for 6 h with DNBS (50 μg/mL, Alfa Aesar) or PMA and ionomycin (50 ng/mL and 1 μg/mL), with the addition of brefeldin A (5 μg/mL, BioLegend) for the last 4 h. Intracellular staining for GzmB and IL-10 was performed using Fixation Buffer and 10× Permeabilization Wash Buffer (BioLegend), as described in the manufacturer’s protocol.
6
Cytokine Profiling of Dendritic Cells
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Dendritic cells were analysed for cytokine production by intracellular cytokine staining. In brief, Cells washed twice with cold PBS, stained for specific surface markers (FITC-conjugated anti-CD11c), fixed (fixation buffer, Bio Legend, USA) and permeabilized (10% permeabilization wash buffer, BioLegend, USA). Cells were then stained for intracellular cytokines with APC-conjugated anti-IL-12p70, and PE-conjugated anti-IL-10. Excess antibodies were washed off through two washing steps and the final pellet was dislodged in 2% formaldehyde and acquired by flow cytometry.
7
Multiparametric Flow Cytometry for Apoptosis
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Cells were harvested, separated by centrifugation, and stained with Fixable Viability Dye eFluorTM 780 (eBioScience, Thermo Fisher), a dye labelling dead cells. After centrifugation, cells were resuspended in phosphate-buffered saline (PBS, Sigma-Aldrich) and fixed using fixation buffer (BioLegend, San Diego, CA, USA). Centrifugation and permeabilization in permeabilization wash buffer (BioLegend) were followed by staining with antibodies to cleaved caspase 3 (Alexa Fluor 647 conjugated, Cell Signaling Technology, Danvers, MA, USA), caspase 8 (unconjugated, Abcam, Cambridge, UK), and cleaved caspase 9 (PE conjugated, Cell Signaling Technology). Cells were separated by another centrifugation step and were afterwards stained with an Alexa Fluor 405-conjugated secondary antibody (Life Technologies, Thermo Fisher Scientific). After centrifugation, cells were resuspended in PBS containing 1% human serum (Biochrom GmbH) and specimens were read on a FACSCanto™ II flow cytometer (BD Biosciences). A minimum of 10,000 events were acquired and analyzed with FACSDiva v6.1.3 software (BD Biosciences). For viability analysis, all events were included and viability dye positive cells were considered dead. The exact gating strategy for caspase analysis is described in Additional file 1 . Experiments were repeated three times (n = 3).
8
Senescence and Blimp1+ Cell Analysis by FACS
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FACS analysis of senescence was performed using Quantitative Cellular Senescence Assay Kit (Cell Biolabs, San Diego, CA, USA) as the manufacturer's protocol. Briefly, preparation of single cells from dorsal skin was through treating minced skin tissues with trypsin and collagenase followed by filtration using a 70 μm mesh filter. Cells were incubated with pretreatment solution at 37 °C for 2 h, subsequently applied to SA-β-Gal substrate solution at 37 °C for 4 h with gentle agitation. After washing stained cells three times with PBS, FACS analysis was performed in the PBS with 1% FBS on a LSRII flow cytometer (BD Bioscience, San Jose, CA, USA) and analyzed using FlowJo software (TreeStar, Ashland, OR, USA) as described.1 (link) For FACS analysis of Blimp1+ cells, after subcutis was removed with scalpel, epidermis was separated from mouse's dorsal skin by incubating overnight with trypsin. Epidermis was further processed to single cells by pipetting with trypsin and filtering through 40 μm mesh filter. For intracellular staining of Blimp1, fixation/permeabilization buffer (Assay Designs, Ann Arbor, MI, USA) and permeabilization wash buffer (Biolegend, San Diego, CA, USA) were used as the manufacturer's instruction. Staining with Blimp1 antibody was followed by staining with secondary antibodies conjugated with Alexa-Fluor 568.
9
Intracellular Cytokine Staining Assay
10
Quantifying Cell Division Dynamics
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In order to assess the cellular division rate, 0.5x103 cells in 100 µl of fully supplemented cell culture medium were seeded per well of a 96-well flat-bottom plate. Cells were fixed every 24 h using fixation buffer (BioLegend, The Netherlands) until 96 h after initial cell seeding. After permeabilization (Permeabilization Wash Buffer; BioLegend, The Netherlands), cells were stained with 10 µM of DAPI. Total cell counts were evaluated using high content imaging (Operetta CLS). Images were acquired using a 10x Air objective (NA = 0.3) in twelve fields of view per well in each of three experiments per condition. Image acquisition parameters and algorithm-based analysis were performed using Harmony 4.9 software (PerkinElmer, Germany).
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