Influenza A Virus challenge: Cells were exposed to PR8-GFP (Influenza A/PR/8/34 (PR8) virus (H1N1), generously provided by Adolfo Garcia-Sastre) overnight at indicated dilutions from stock. Following the incubation period, cells were fixed and permeabilized with the BD Fixation/Permeabilization kit (BD). Cells were fixed for 20 min at 4°C and stained with DAPI (1:10,000 in 1XPerm/Wash buffer) for 20 min at RT. Cells were then washed and left in PBS. DAPI and GFP signals were acquired on a Celigo Imaging Cytometer (Nexcelom) and quantified in CellProfiler (McQuin et al., 2018). Experiments were carried out with n=3 biological replicates. Zika Virus challenge: Cells were exposed to ZIKV (strain PRVABC59, GenBank: KU501215.1) for one hour, washed three times, and allowed to rest overnight. Supernatants from these cells were then harvested and diluted at 1:100 dilution followed by 1:3 serial dilutions and placed on previously plated Veros (ATCC). After 7 days, Veros were fixed and permeabilized with the BD Fixation/Permeabilization kit (BD). Cells were fixed for 20 min at 4°C and stained with anti-Flavivirus Group Antigen 4G2, 1:1000 (Millipore) for 1 hour at RT. Cells were then washed and stained with secondary (1:10,000) and DAPI for 45 minutes at RT. Cells were then washed and DAPI and AF647 signals were acquired on a Celigo Imaging Cytometer (Nexcelom).
Fixation permeabilization kit
Manufactured by BD
Sourced in United States, Canada
The Fixation/Permeabilization Kit is a laboratory product that prepares samples for analysis by fixing and permeabilizing cells. It facilitates the access of antibodies or other detection reagents to intracellular targets. The kit provides the necessary solutions and buffers to perform these critical sample preparation steps.
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Lab products found in correlation
Facscanto 2, by BD (15 mentions)
Ionomycin, by Merck Group (14 mentions)
Golgiplug, by BD (14 mentions)
Golgistop, by BD (13 mentions)
Lsrfortessa, by BD (10 mentions)
Facsaria 2, by BD (9 mentions)
Celltrace violet, by Thermo Fisher Scientific (8 mentions)
Brefeldin a, by Merck Group (7 mentions)
Phorbol myristate acetate (pma), by Merck Group (7 mentions)
Facscalibur, by BD (7 mentions)
Facscanto 2 flow cytometer, by BD (6 mentions)
Brefeldin a, by BD (5 mentions)
Fc block, by BD (5 mentions)
Brefeldin a, by BioLegend (5 mentions)
Near ir dead cell stain kit, by Thermo Fisher Scientific (5 mentions)
Cell stimulation cocktail, by Thermo Fisher Scientific (5 mentions)
Transcription factor buffer set, by BD (5 mentions)
Perm wash buffer, by BD (5 mentions)
Leukocyte activation cocktail, by BD (4 mentions)
Lsm 880 confocal microscope, by Zeiss (4 mentions)
198 protocols using fixation permeabilization kit
1
Influenza and Zika Virus Challenges
2
Multimodal Characterization of Intestinal Organoids
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Human intestinal organoids were harvested using Cell Recovery Solution (Corning) from Matrigel. The organoids were treated with a blocking solution (3% bovine serum albumin solution in PBS). They were then fixed and permeabilized using Fixation/Permeabilization Kit (BD Biosciences), after which they were stained with BODIPY 493/503 (Thermo Fischer Scientific), Alexa Fluor 568 Phalloidin (Thermo Fischer Scientific), and DAPI (Dojindo) according to the manufacturers’ instructions. BODIPY, Phalloidin, and DAPI were used to counterstain neutral lipids, F-actin, and DNA, respectively.
3
Flow Cytometric Analysis of Immune Cell Phenotypes
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The cells were either directly labelled with the above-noted antibodies using PBS with 2.5% FCS for staining and washes or were fixed, permeabilized and then stained using the Fixation/Permeabilization kit (BD Biosciences, Stockholm, Sweden) following the manufacturer’s protocol. To reduce non-specific antibody binding, the FcR Blocking Reagent (human or murine) of Miltenyi Biotech was used prior to staining. Antibodies were added to the samples (2 μg in 50 μL) for 30 min at 4 °C, then the samples were washed and incubated with APC-labeled anti-human IgG (BD Biosciences) for 30 min at 4 °C. After two subsequent washes, the samples were fixed in 1% paraformaldehyde and stored at 4 °C prior to analysis. To evaluate neutrophil death, we used Live/Dead fixable dead cell staining (Thermo Fisher), and in some experiments SYTOX Green (Thermo Fisher) or BV421-labeled Annexin-V (BD Biosciences). We studied degranulation using AlexaFluor 488-labelled anti-CD107a (Biolegend, San Diego, CA, USA). The cells were analyzed on a FACSVerse flow cytometer, with a gating strategy depicted on Supplementary Figure S2 , and the data were analyzed using FlowJo software v10.8.1(BD Biosciences).
4
Comprehensive Analysis of B Cell Activation
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Human PMBC were plated in 96-well U bottom plates (3 × 105/well) and divided into three groups: (1) control group; (2) R848 group (2.5 μg/ml); (3) CpG group (5 μg/ml). Cells were cultured for 3 days, then washed and stained with the following panels: B cell activation marker panel (CD19-FITC, CD80-PE, CD86-APC, CD40-APCH7, and HLA-DR-PE-Cy7); antibody panel (CD19-FITC, IgM-PE, and IgG-BV605); cytokine panel (CD19-FITC, IL-6-PE, and IL-10-APC); and isotype switch marker panel (CD19-FITC, CD27-PE-Cy7, and IgD-Percp-Cy5.5). For the intracellular staining panel, golgi plug (BD, USA) was added 5 h before harvesting the cells. After staining the surface marker, we used the Fixation/Permeabilization Kit (BD, USA) for intracellular marker staining. Flow cytometric analysis was performed using a Canto II flow cytometer (BD, USA). CS&T beads (BD, USA) were used in every experiment to maintain quality performance over time, Compensation was performed by using the negative control sample, single positive controls, and compensation beads (BD, USA) (for low expression markers). Flow cytometry data were analyzed using Flowjo 10.4 software (Treestar, USA).
5
NK92MI Cytokine Production Assay
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NK92MI cells (5×105) were stimulated with PMA and Ionomycin (Sigma) for one hour at 37°C in 5% CO2. Brefeldin A (BD Biosciences) was then added to the cultures, which were incubated for five more hours. After six hours of total incubation, the cells were centrifuged and resuspended in 50μL of staining buffer (phosphate-buffered saline supplemented with 2% fetal bovine serum) followed by immunostaining with fluorochrome-conjugated mAbs for surface markers. Cells were washed, fixed with Fixation/Permeabilization Kit (BD Biosciences), and intracellularly stained with fluorochrome-conjugated mAbs to TNF-α and IFN-γ. Finally, cells were washed and analyzed on a Fortessa flow cytometer (BD Biosciences). Data analysis was performed using FlowJo 7.6 software (TreeStar).
6
Immunophenotyping of ADEPCs and ADSCs
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ADEPCs and ADSCs were trypsinized, washed three times with PBS containing 1% BSA, and pelleted by centrifugation at 400 g. Cells were incubated with membranous (CD14, CD31, CD34, CD45 and VEGF Recepter-2 (VEGFR-2)) and cytoplasmic (Stro-1, eNOS, and α-SMA) antibodies at RT for 40 min. A fixation/permeabilization kit (BD Biosciences) was applied for staining intracellular antigens. Cells were then washed twice and incubated at RT for 30 min with Alexa Fluor 488-conjugated goat anti-mouse secondary antibody in the dark. The labeled cells were washed twice, resuspended and finally analyzed with FACSCaliber (BD Biosciences). An isotype-matched IgG was set as the control for each primary antibody. The flow cytometric analysis was repeated six times.
7
Multiparameter Flow Cytometry Analysis
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The antibodies used for FACS analysis are summarized in Table 1 . The efluor 450-labeled anti-Foxp3 staining was performed using the eBioscience kit and protocol. The fixation/permeabilization kit (BD Biosciences) was used for intra-cellular staining of active caspase-3. Cell proliferation dye 450 (ebioscience) was used to help exclude APCs in the in vivo experiment. Flow cytometry events were acquired on a FACSCanto II flow cytometer (BD Biosciences) and analyzed using FlowJo (Tree Star, Ashland, OR, USA) software.
8
Flow Cytometry Cell Staining Protocol
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Fluorochrome-conjugated monoclonal antibodies against rat or human molecules for flow cytometry staining (antibody clone, fluorochrome, supplier, and catalog number) are listed in Additional file 1 : Table S3. Fixable viability stain 620 (FVS-620, 564,996, BD Biosciences) was used to discriminate between live and dead cells, which were then blocked with Fc-block (553,142, BD Biosciences, Franklin Lakes, NJ, USA) and stained with antibodies. Intracellular cytokine (CD206, Proteintech, Rosemont, IL, USA) staining was performed using a Fixation/Permeabilization Kit (BD Biosciences). The data were acquired using a NovoCyte Flow Cytometer.
9
Tumor Dissociation and Analysis
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The tumors were harvested on the sixth day after the last administration, minced, and digested in RPMI-1640 medium containing collagenase IV (0.1%, Gibco), nuclease, and 1% fetal bovine serum at 37 °C for 40 ~ 60 min, after which the cell suspensions were filtered. Fixable Viability Stain 620 (FVS620, 564996, BD Biosciences) was used to discriminate live or dead cells; the cells were then blocked with Fc-block (553142, BD Biosciences) and stained with antibodies. Nuclear factors were permeabilized using a FoxP3 Fixation and Permeabilization Kit (00-5521-00, Invitrogen), while intracellular cytokines were permeabilized using a Fixation/Permeabilization Kit (554714, BD Biosciences) and detected with antibodies. Data were acquired on a NovoCyte flow cytometer. A representative flow gating scheme was shown in Figure S11 .
Detailed antibodies, apoptosis assay, western blot, R-T PCR, immunofluorescence, analysis of HMGB1 and ATP release, immunohistochemical staining, and T cell depletion assay are described in theSupplementary materials and methods.
Detailed antibodies, apoptosis assay, western blot, R-T PCR, immunofluorescence, analysis of HMGB1 and ATP release, immunohistochemical staining, and T cell depletion assay are described in the
10
Intracellular Cytokine and Transcription Factor Staining
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The cells were stained with conjugated antibodies (Abs) and permeabilized with a Fixation/Permeabilization Kit according to the manufacturer’s instructions (BD Biosciences, San Diego, CA, USA), before being stained with Abs to IL-17, TGF-β1, and Foxp3.
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