To stain the CD8+ T cells specific to the Kb-restricted HBV Env190–197 and Cor93–100 epitopes, recombinant soluble dimeric mouse H-2K[b]: Ig fusion proteins (DimerX I, BD Bioscience) were loaded with the respective peptides overnight and then used to stain mouse lymphocytes according to the technical instructions. The cells were first incubated with CD16/CD32 rat anti-mouse antibody (clone 2.4G2; BD Pharmingen) to block FcRs. Then, the cells were stained with anti-CD8, 7AAD and anti-PD-1 (clone J43; BD Pharmingen). After washing, dimer staining was performed by incubating the dimer and cells for 1.5 h at 4 °C. The cells were then washed and incubated with an anti-IgG1 antibody (clone 85.1; eBioscience) for 30 min at 4 °C. The stained samples were run on a FACSCalibur (Becton Dickinson) or NAVIOS Flow Cytometer (Beckman Coulter GmbH). The data were analyzed using FlowJo software. The percentage of specific CD8+ T cells in the liver was calculated based on the percentage of dimer+ CD8+ T cells within the CD8+ T-cell population of viable lymphocytes recovered from each liver.
Cd16 cd32 rat anti mouse antibody clone 2.4g2
Manufactured by BD
Sourced in Switzerland
The CD16/CD32 rat anti-mouse antibody (clone 2.4G2) is a laboratory reagent used for blocking Fc receptors in immunoassays and flow cytometry experiments involving mouse samples.
Automatically generated - may contain errors
Lab products found in correlation
Dimerx 1, by BD (2 mentions)
Anti igg1 antibody clone 85.1, by Thermo Fisher Scientific (2 mentions)
Facscalibur, by BD (1 mentions)
Navios flow cytometer, by Beckman Coulter (1 mentions)
Facsdiva software, by BD (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Lsr fortessa, by Tree Star (1 mentions)
Canto 2, by BD (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Fitc pna, by Merck Group (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Influx cell sorter, by BD (1 mentions)
4 protocols using cd16 cd32 rat anti mouse antibody clone 2.4g2
1
Murine Hepatitis B-Specific CD8+ T Cell Staining
2
Characterization of Antigen-Specific CD8+ T Cells
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Surface and intracellular staining were performed as described previously [18 (link), 40 (link)]. The gating strategy for Tregs is shown in S1 Fig . For staining CD8+ T cells specific to the Kb-HBV Cor93-100 (MGLKFRQL), recombinant soluble dimeric mouse H-2K[b]:Ig fusion protein (DimerX I, BD Bioscience) were loaded with HBV Cor93-100 for over night, and then used to stain mouse lymphocytes according to the technical instruction. The cells were incubated with CD16/CD32 rat anti-mouse antibody (clone 2.4G2; BD Pharmingen) to block FcRs. After washing, dimer staining was performed by incubation of dimer and cells for 1.5 hours at 4°C. The cells were washed and incubated with anti-IgG1 antibody (clone 85.1; eBioscience) for 30 minutes at 4°C. Data were acquired on a LSR II flow cytometer (Becton Dickinson) from 350,000–500,000 lymphocyte-gated events per sample. Analyses were done using FACSDiva software (Becton Dickinson) and FlowJo software (Treestar).
3
Multicolor Flow Cytometry Staining
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Single-cell suspensions from tissues and cells from BAL fluid were stained for surface markers for 30 min at 4°C in PBS containing 0.01% (m/v) sodium azide and 1% (m/v) BSA. Propidium iodide (0.3 μg/mL, Sigma, St. Louis, MO, USA) in PBS was used to distinguish vital cells. Samples were treated with rat anti-mouse CD16/CD32 antibody (clone 2.4G2, BD Biosciences, Basel, Switzerland) to block FcR-mediated non-specific antibody binding prior to incubation with fluorochrome-conjugated antibodies. Acquisitions were made with a BD Canto II or LSR Fortessa and analyzed using FlowJo software (version 10.0.7, Treestar).
4
Flow Cytometry Characterization of B-cell Subsets
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Peritoneal washout cells and splenocytes were obtained from 8- to 14-week-old wild-type (WT) or AID knock out (KO) mice and were stained with fluorescence-labeled antibodies to B220, CD5, CD25, CD23, and GL-7 and with peanut agglutinin (PNA). B-cell populations (peritoneal B-1a cells: B220lo/CD5+, peritoneal CD25+ B-1a cells: B220lo/CD5+CD25+, peritoneal CD25− B-1a cells: B220lo/CD5+CD25−, splenic B2 cells: B220hiCD5−CD23+, or GC B cells: B220+/GL-7+/PNAhigh) were isolated using the Influx cell sorter (BD Biosciences). Post-sort, reanalysis of the B-cell populations showed them to be ≥98% pure. Cells were blocked with rat anti-mouse CD16/CD32 antibody (clone 2.4G2), stained with immunofluorescent antibodies, and then analyzed on a FACSCalibur flow cytometer (BD Biosciences) with appropriate gating. Images were constructed with FlowJo 6.0 software (Tree Star). PE-conjugated rat anti-mouse CD25 (clone PC61) was obtained from BD Pharmingen. The following antibodies were obtained from Biolegend: perCP-Cy5.5-conjugated rat anti-mouse CD45R/B220 (clone RA3-6B2); Alexa 647-conjugated rat anti-mouse CD5 (clone 53-7.3); and Alexa 647-conjugated rat anti-mouse GL-7. FITC-PNA was obtained from Sigma. CD23-PE-Cy7 (clone 2G8) was obtained from Abcam.
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