Cells were dissociated with Accutase for 30 min and then washed in Flow buffer (DMEM, 2% FBS, and 1mM L-glutamine). Cells were centrifuged at 200 g for 4 min, resuspended in cold PBS, counted, and diluted to a concentration of 1x106 cells/100μL. Cells were then centrifuged and resuspended in 300 μL BD Cytofix/Cytoperm (BD Biosciences) buffer and incubated on ice for 30 min. Cells were centrifuged for 4 min at 2,000 RPM and resuspended in 600 μL of cold BD Permeabilization/Wash buffer (BD Biosciences). 30 μL goat serum was added followed by incubation on ice for 30 min. Cells were divided in 3: unstained control, secondary antibody control, and sample to stain (200 μL each). All tubes were centrifuged for 4 min at 2,000 RPM and the cells were resuspended in 200 μL of Antibody buffer: BD perm/wash buffer + 10 μL goat serum with or without BRN3A antibody (1:100). Cells were incubated o/n at 4°C. Cells were then washed twice with 300 μL BD Permeabilization/Wash buffer, resuspended in Antibody buffer with or without AF488 goat-anti-mouse (1:500), and incubated on ice for 30 min. Cells were then washed 3X with BD perm/wash buffer. Cells were filtered and analyzed using a Cytoflex S (Beckman). Analysis was done using FlowJo.
Permeabilization wash buffer
Manufactured by BD
Sourced in United Kingdom, United States
Permeabilization/wash buffer is a laboratory reagent used to facilitate the permeabilization and washing of cells or tissue samples during various analytical procedures. It is designed to create temporary pores in cell membranes, allowing for the introduction or removal of specific molecules or compounds. The buffer's core function is to enable the effective permeabilization and washing of samples, which is a crucial step in many cell-based assays and immunological techniques.
Automatically generated - may contain errors
Lab products found in correlation
Fixation permeabilization solution, by BD (6 mentions)
Fixation permeabilization buffer, by BD (3 mentions)
Fc block, by BD (3 mentions)
Il 1β pe, by Thermo Fisher Scientific (3 mentions)
Tnf α pe cy7, by Thermo Fisher Scientific (3 mentions)
Il 10 apc and il 4 percp cy5, by BioLegend (2 mentions)
Fixation buffer, by BD (2 mentions)
Facscanto 2 flow cytometer, by BD (2 mentions)
Golgiplug, by BD (2 mentions)
Live dead aqua, by Thermo Fisher Scientific (2 mentions)
Fitc anti mouse cd8, by BD (2 mentions)
Anti mouse cd4 pe, by BD (2 mentions)
Golgiplug containing brefeldin a, by BD (2 mentions)
Il 1β fitc, by Thermo Fisher Scientific (2 mentions)
Tnf pe cy7, by Thermo Fisher Scientific (2 mentions)
Cytofix cytoperm buffer, by BD (2 mentions)
Permeabilization wash buffer, by BioLegend (2 mentions)
Cytexpert software, by Beckman Coulter (2 mentions)
Cytoflex flow cytometer, by Beckman Coulter (2 mentions)
Cytofix buffer, by BD (2 mentions)
26 protocols using permeabilization wash buffer
1
Flow Cytometric Analysis of BRN3A Expression
2
Multiparametric Flow Cytometry Analysis
3
Ex vivo intracellular cytokine staining
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For intracellular cytokine staining, an ex vivo brefeldin A protocol was followed. Prior to staining, brain leukocytes were incubated with BFA (10 μg/mL, Sigma) in 1 mL complete RPMI for 2 h at 37 °C (5% CO2). Afterward, cells were resuspended in Fc Block, stained for surface antigens and washed in 100 μL of fixation/permeabilization solution (BD Biosciences) for 20 min. Cells were then washed twice in 300 μL permeabilization/wash buffer (BD Biosciences), resuspended in an intracellular antibody cocktail (0.25 μg for each antibody, 1:100 dilution) containing TNFα‐PE‐Cy7 (eBioscience) and IL‐1β‐PE (eBioscience), IL‐10‐APC and IL‐4‐PerCP‐Cy5.5 (BioLegend) and subsequently fixed.
4
Macrophage LAMP-1 Assay by Flow Cytometry
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At the specified time point (between 2 and 24 h) postinfection, macrophages were harvested, transferred to 200 µl fixative-permeabilization buffer (BD Biosciences) and incubated for 30 min at room temperature on a roller. Following incubation, the sample volume was increased to 1 ml with 800 µl of 1× permeabilization-wash buffer (BD Biosciences) and centrifuged for 5 min at 300 × g. Following centrifugation, cells were resuspended into 200 µl permeabilization-wash buffer (BD Biosciences) containing 3.5 µg · ml−1 PE/Dazzle 594 anti-mouse CD107a (LAMP-1) antibody (BioLegend, UK) and incubated for 1 h at room temperature on a roller. Following incubation, 800 µl of PBS (Gibco) was added to the samples and centrifuged for 5 min at 300 × g. Finally, all cell pellets were resuspended into 50 µl PBS (Gibco) for analysis by imaging flow cytometry.
5
F-Actin Staining of Activated NK Cells
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For F-actin staining, IL-2 activated NK cells were serum and cytokine-starved for 4 h. The cells were then left untreated or treated with 10 ng/ml recombinant mouse CCL2/MCP-1 (R&D Systems) for 30 minutes at 37°C. The cells were fixed in suspension with Fixation buffer (BD Biosciences) and 2.5 x 105 cells were cytospun onto microscope slides (Shandon). The slides were then permeabilized with Permeabilization/Wash buffer (BD Biosciences) and stained with rhodamine-conjugated Phalloidin (Invitrogen) followed by mounting with Fluorescent Mounting Media (Vector Labs). The cells were visualized by confocal microscopy with a 63x oil objective on a Leica TCS SP5 Laser Scanning Confocal Microscope, and captured images were analyzed with LAS-AF software. Definiens Tissue Studio version 4.7 was used to analyze the images for green fluorescence intensity. First a nucleus detection algorithm was applied to the DAPI channel to segment nuclei based on intensity and size thresholds. Next a simple growth algorithm of 1 micron was applied to generate a cytoplasm around each nuclei. The intensity for green fluorescence was measure in each cell, nucleus, and cytoplasm.
6
Quantifying Peptide-Specific Cytotoxic T-Cells
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Peptide-specific cytotoxic T-cells were quantified by flow cytometry analysis. Single cell suspensions of excised spleens were obtained using 70 μm cell strainers (BD Falcon). Subsequently, 2*106 splenocytes were plated per well in 48-wells culture plates (Greiner) and restimulated with 50 ng M158–66 peptide for 18 h at 37°C 5% CO2. Next, Golgi-plug (1:1000, BD) was added to the cells to inhibit cellular protein and cytokine transport, and cells were incubated for another 4 h. Subsequently, cells were transferred to a 96-wells plate, washed twice with FACS buffer (PBS, 0.5% BSA), and stained with anti-mouse CD4-PE (BD Biosciences), anti-mouse CD8-FITC (BD Biosciences) and Live/dead-Aqua (Invitrogen). Next, cells were washed twice with FACS buffer, and fixed with fixation-permeabilization buffer (BD Biosciences). Subsequently cells were washed with permeabilization wash buffer (BD Biosciences), and intracellular staining was performed with IFN-γ-APC (Biolegend). Finally, cells were washed with FACS buffer and 1.5 to 2 million cells were measured on a FACS Canto II flow cytometer (BD Biosciences). Data was analyzed using FlowJo software version 9 (Tree Star) for Mac OSX.
7
In Vivo Cytokine Staining Protocol
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For intracellular cytokine staining, an in vivo brefeldin A (BFA) protocol was followed. Briefly, 10 mL/kg of BFA (Sigma, 0.5 mg/mL in DMSO) was injected via tail vein. Ten hours later, animals were sacrificed and tissue was harvested as noted above. Prior to staining, 1 ul of GolgiPlug containing brefeldin A (BD Biosciences) was added to 800 ul complete RPMI and cells were incubated for 2 h at 37 °C (5 % CO2). Afterward, cells were re-suspended in Fc Block, stained for surface antigens and washed in 100 ul of fixation/permeabilization solution (BD Biosciences) for 20 min. Microglia were then washed twice in 300 ul permeabilization/wash buffer (BD Biosciences), re-suspended in an intracellular antibody cocktail containing TNF-PE-Cy7 (eBioscience) and IL-1β-FITC (eBioscience) and subsequently fixed (N = 5-7/group).
8
Profiling CAR-T Cell Metabolic Markers
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CAR-T cells (0.1 × 106) were cocultured with 0.1 × 105 HCC827-Luc tumor cells in 96-well plates in duplicate or triplicate as described above. After 72 hours, CAR-T cells were collected and stained with a fixable viability dye (Zombie NIR or ghost violet 510) and fluorophore-conjugated CD3 and EGFR as described above. CAR-T cells were fixed and permeabilized in fixation/permeabilization solution (BD Biosciences) according to the manufacturer’s instructions; washed with permeabilization/wash buffer (BD Biosciences) according to the manufacturer’s instructions; and incubated in unconjugated primary rabbit anti-human MCT4, HK1, ALDOA, or PFKFB3 for 15 to 30 min at room temperature. Afterward, the CAR-T cells were washed with permeabilization/wash buffer and incubated in fluorophore-conjugated anti-rabbit antibody for 15 to 30 min at room temperature. Cells were washed in permeabilization/wash buffer and analyzed using a Cytek Aurora flow cytometer.
9
Intracellular Cytokine Profiling of Brain Leukocytes
10
Flow Cytometry Analysis of MER Expression
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Sub-confluent cultures (approximately 5 × 105 cells) were washed with PBS and lifted with 0.02% EDTA in PBS. Harvested cells were fixed in 4% paraformaldehyde (Figure 2C ) or not (Figure 1 ), then washed in FACS wash buffer (2% FBS and 0.02% azide in PBS) prior to staining in 50 μl staining solution (1% FBS and 0.02% azide in PBS) containing primary antibody, murine immunoglobulin (mIgG1, R&D Systems, MAB002), or vehicle control, for 15-30 minutes at 4°C. Cells were washed again in FACS wash buffer, and then incubated in staining solution containing fluorophore-conjugated secondary antibody or vehicle control for 15-30 minutes at 4°C. Stained cells were washed in FACS wash buffer, resuspended in staining solution, and fluorescence of surface-bound antibodies was measured by flow cytometry. For assessment of total MER, cells were fixed in 4% paraformaldehyde, washed in FACS wash buffer, permeabilized in permeabilization/wash buffer (BD, 554723), resuspended in staining solution containing secondary antibody or vehicle control for 30 minutes at 4°C, washed in perm/wash buffer, and resuspended in staining solution before analysis by flow cytometry.
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