α ifnγ apc

Manufactured by BD

Sourced in United States

α-IFNγ-APC is a fluorescently-labeled antibody that binds to the cytokine interferon-gamma (IFNγ). It is used in flow cytometry applications to detect and quantify IFNγ-producing cells.

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3 protocols using α ifnγ apc

Comprehensive Immune Profiling by FACS

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Cell suspensions were stained for FACS analysis with the following antibodies: α-CD3-PE (BD Biosciences), α-CD8-BV605 (BD Biosciences), α-CD45-PercP (BD Biosciences), α-CD45-PE (BD Biosciences), α-CD11b-FITC (BioLegend), α-CD11c-FITC (BioLegend), α-CTLA4-PE (BD Biosciences), α-granzyme B-Alexa fluor 647 (BD Biosciences), α-IFNγ-APC (BD Biosciences), α-KI67-BV421 (BD Biosciences), α-TIM3-PE (eBioscience), α-TIGIT-PE (BD Biosciences), α- TNFα-PE (BD Biosciences), α-IL17r-PE (BD Biosciences), and the corresponding isotype control (mouse IgG1, Southern Biotech). The live/dead fixable near-infrared dye (Invitrogen) was used to exclude dead cells. Intracellular fixation and permeabilization buffers from eBioscience were used for cytokine staining.
Acquisition was performed on FACS BD Fortessa and the dedicated software CellQuest (BD Biosciences). Data was analyzed with FlowJo 7.5.5 software (TreeStar Inc).

D’Amico L., Menzel U., Prummer M., Müller P., Buchi M., Kashyap A., Haessler U., Yermanos A., Gébleux R., Briendl M., Hell T., Wolter F.I., Beerli R.R., Truxova I., Radek Š., Vlajnic T., Grawunder U., Reddy S, & Zippelius A. (2019). A novel anti-HER2 anthracycline-based antibody-drug conjugate induces adaptive anti-tumor immunity and potentiates PD-1 blockade in breast cancer. Journal for Immunotherapy of Cancer, 7, 16.

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Expansion and Characterization of HIV-Specific CD8+ T Cells

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B-EBV lines were generated for each donor by culturing PBMCs in RPMI, 20% fetal bovine serum (FBS), 20nM FK506 (AG Scientific), and Ebstein-barr virus (EBV)-containing supernatant from the virus-producing B95.8 marmoset cell line (ATCC) at an MOI of 100. B-EBV cell lines were then loaded with 0.5–5 mg/mL HIV Clade AE peptide pools overnight. Peptide pools were made with 20 15-mer peptides per pool of HIV PTE and HIV Consensus A peptides (gag, pol, nef, env) obtained through the AIDS Reagent Program, Division of AIDS, NIAID. Expanded primary CD8+ T cells were added to the loaded B-EBV (2:1), co-stimulated with 1 μg/μL αCD28/CD49d (BD Biosciences) and incubated for 12 hours with GolgiPlug protein transport inhibitor (BD Biosciences). After incubation, cells were stained with αCD8-FITC, αCD3-PacificBlue (BD Biosciences), and αCD20-PECy7 (BioLegend) prior to fixation/permeabilization and intracellular staining with αIFNγ-APC (BD Biosciences). Live/dead stain with Vivid-amcyan was used to exclude dead cells from the analysis. The T-cell receptor Vbeta repertoire was analyzed by flow cytometry using the IOTest^® Beta mark (Beckman Coulter) in conjunction with αCD8-Pacific Blue (BD Biosciences). Stained CD8+ T cells were run on an LSRII flow cytometer using DiVA software (BD Biosciences) and analyzed with FlowJo (Treestar).

Kessing C.F., Spudich S., Valcour V., Cartwright P., Chalermchai T., Fletcher J.L., Nichols C., Josey B.J., Slike B., Krebs S.J., Sailsuta N., Lerdlum S., Jagodzinski L., Tipsuk S., Suttichom D., Rattanamanee S., Zetterberg H., Hellmuth J., Phanuphak N., Robb M.L., Michael N.L., Ananworanich J, & Trautmann L. (2017). High Number of Activated CD8+ T Cells Targeting HIV Antigens are Present in Cerebrospinal Fluid in Acute HIV Infection. Journal of acquired immune deficiency syndromes (1999), 75(1), 108-117.

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ESAT-6 Peptide Library Staining Protocol

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Monoclonal antibodies α-CD3-FITC (BD Pharmingen 553062), α-CD4-PerCP (BD Pharmingen 553052), and α-IFNγ-APC (BD Pharmingen 554413) were obtained from BD Biosciences (San Jose, California, USA). Intracellular staining was performed using the BD permeabilization kit Cytofix/Cytoperm—Permwash (BD Biosciences 554714). CFSE (21888) and PKH26 (PKH26-GL) fluorochrome labels were purchased from Sigma-Aldrich (Saint Louis, MO, USA) and cell staining was performed as suggested by manufacturers. BFA was obtained from Sigma-Aldrich (St. Louis, MO), and PowerBlock reagent from Biogenex (HK085-5K). ESAT-6 library of overlapping peptides (staggered by 4 amino acids spanning the entire ESAT-6) were synthesized by the Proteomics Resource Center (The Rockefeller University). The ESAT-6 peptide library was resuspended at 1 mg/ml of each peptide in 100% DMSO, the library was divided into three pools of 9–8 peptides each, spanning amino acids 1–36 (pool 1), amino acids 25–65 (pool 2), and amino acids 57–96 (pool 3) (Fig 1B).

Silva-Sánchez A., Meza-Pérez S., Flores-Langarica A., Donis-Maturano L., Estrada-García I., Calderón-Amador J., Hernández-Pando R., Idoyaga J., Steinman R.M, & Flores-Romo L. (2015). ESAT-6 Targeting to DEC205+ Antigen Presenting Cells Induces Specific-T Cell Responses against ESAT-6 and Reduces Pulmonary Infection with Virulent Mycobacterium tuberculosis. PLoS ONE, 10(4), e0124828.

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