Anti granzymeb pacific blue

Manufactured by BioLegend

Sourced in United States

Anti-granzymeB-Pacific Blue is a fluorochrome-conjugated antibody that binds to human granzyme B. Granzyme B is a serine protease that plays a key role in cell-mediated cytotoxicity. This antibody can be used for the detection and analysis of granzyme B-expressing cells by flow cytometry.

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Lab products found in correlation

Bv510 streptavidin, by BioLegend (2 mentions) Anti cd8 fitc clone sk1, by BioLegend (2 mentions) Anti cd62l pe cy5 clone dreg 56, by BioLegend (2 mentions) Anti cd3 af700 clonehit3a, by BioLegend (2 mentions) Anti cd45ra bv605, by BioLegend (2 mentions) Anti granzyme a percp cy5, by BioLegend (2 mentions) Bv650 streptavidin, by R&D Systems (2 mentions) Anti granzyme k pe, by BioLegend (2 mentions) Anti granzyme m efluor660, by Thermo Fisher Scientific (2 mentions) Liberase, by Roche (1 mentions) Histopaque 1077, by Merck Group (1 mentions) Pharm lyse, by BD (1 mentions) Anti cd3 pe, by BD (1 mentions) Anti mouse cd16 cd32 clone 2.4g2, by BD (1 mentions)

4 protocols using anti granzymeb pacific blue

Isolation and Analysis of Tumor-Infiltrating Lymphocytes

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Tumors were harvested and tissues digested with 250 µg/mL of Liberase (Roche, Basel, Switzerland) and incubated for 30 min at 37 °C with shaking at 105 rpm. Fetal bovine solution was added to stop the digestion reaction, samples were filtered, and TILs were enriched by using Histopaque 1077 (Sigma, St. Louis, MO, USA, Cat. #H8889). Cells were then blocked with anti-CD16/CD32 before being stained for flow cytometry. Stains included fluorochrome-conjugated anti-CD4 BV510 (Cat. #100449), anti-CD8 PercpCy5.5 (Cat. #100734), anti-CD45 Pacific blue (Cat. #103126), anti-CD49b FITC (Cat. #108905), anti-CD44 APC (Cat. #103012), anti-CD11c PE (Cat. #117308), anti-CD103 PE-Cy7 (Cat. #121426), anti-B220 FITC (Cat. #103206), anti-PDCA-1 APC (Cat. #127016), anti-IFNg Alexa 488 (Cat. #505813), and anti-Granzyme B Pacific blue (Cat. #515407), all from BioLegend (San Diego, CA, USA). Flow cytometry data were analyzed with FlowJo software (Ashland, OR, USA).

Younes A.I., Barsoumian H.B., Sezen D., Verma V., Patel R., Wasley M., Hu Y., Dunn J.D., He K., Chen D., Menon H., Masrorpour F., Gu M., Yang L., Puebla-Osorio N., Cortez M.A, & Welsh J.W. (2020). Addition of TLR9 agonist immunotherapy to radiation improves systemic antitumor activity. Translational Oncology, 14(2), 100983.

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Isolation and Phenotyping of Tumor-Infiltrating Immune Cells

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Tumor tissues (200 mg) were cut into small pieces in 0.5 ml RPM1 640 culture medium. Liberase was added to dissociate the connected tumor cells at room temperature for 15 min. Dissociated cells were passed through a cell strainer (70um). After spinning down the cells at 1000 g centrifugation for 10 min, red blood cells were lysed with 1 ml BD pharm lyse on ice. Cells were collected at 1000 g centrifugation for 10 min. 2 × 10⁶ cells were used for each staining sample. Cells were resuspended in RPM1 640 with 3% FBS. Anti-mouse CD16/CD32 clone 2.4G2 (BD Pharmingen, #553142) was used to block Fc receptors on cells. Total T cells were stained by Anti-CD3-PE (BD Pharmingen, clone 145-2c11, #553064) for 30 min on ice. Fixation/Permeabilized (eBioscience) was used to fix and permeabilize cells. Then activated cytotoxic T cells was further stained with Anti-Granzyme B-pacific blue (BioLegend, clone GB11, #515408) and T regulatory cells were stained with Anti-FOX3P-APC (eBioscience, clone FJK16s, #17-5773-83). The stained cells were washed and analyzed by flow cytometry LSR II (BD Canto II, New Jersey, USA). Details of flow cytometry analysis can be found in our previous report^{38 (link)}.

Yang X., Lam W., Jiang Z., Guan F., Han X., Hu R., Cai W., Cheng W., Liu S.H., Cheng P., Cai Y., Rattray N.J., Johnson C.H., Chen L, & Cheng Y.C. (2021). YIV-906 potentiated anti-PD1 action against hepatocellular carcinoma by enhancing adaptive and innate immunity in the tumor microenvironment. Scientific Reports, 11, 13482.

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Assessing CD8+ T Cell Cytolytic Potential

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Rested ex vivo CD8⁺ T cells were assessed for CMV and
HIV specificity using HLA-matched tetramers. Tetramers were made via
biotinylated monomer conjugation to BV510 streptavidin (BioLegend). Monomers
were obtained from Dr. Soren Buus (University of Copenhagen). The A02-NV9
tetramer was used to stain for CMV-specificity while pools of A02-SL9, B27-KK10,
B53-QW9, B53-YY9, B57-IW9(p24), B57-IW9(RT), B57-KF11, B57-QW9, and B57-TW10
tetramers were used to stain for HIV-specificity. Subsequent surface stains
included anti-CD62L-PE-Cy5 (clone DREG-56; BioLegend), anti-CD3-AF700 (clone
HIT3a; BioLegend), anti-CD8-FITC (clone SK1; BioLegend), anti-CD45RA-BV605
(clone HI100; BioLegend), and LIVE/DEAD Blue. Following permeabilization, the
cells were intracellular stained using anti-perforin PE/Cy7 (clone B-D48;
BioLegend), anti-granzyme A-PerCp/Cy5.5 (clone CB9; BioLegend), anti-granzyme
B-Pacific Blue (clone GB11; BioLegend), anti-granzyme H (biotinylated and
secondary stained with BV650 streptavidin) (polyclonal antibody, R&D),
anti-granzyme K-PE (clone GM26E7; BioLegend), and anti-granzyme M-eFluor660
(clone 4B2G4; eBioscience). The cells were analyzed by flow cytometry.
Naïve CD8⁺ T cells within the mixed population of
CD8⁺ T cells were used as internal gating controls for
perforin and each granzyme as these cells do not express these effector
molecules.

Clayton K.L., Collins D.R., Lengieza J., Ghebremichael M., Dotiwala F., Lieberman J, & Walker B.D. (2018). Resistance of HIV-infected macrophages to CD8 T lymphocyte-mediated killing drives immune activation. Nature immunology, 19(5), 475-486.

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Assessing CD8+ T Cell Cytolytic Potential

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