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Cd8α 53 6

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CD8α (53-6.7) is a monoclonal antibody that binds to the CD8α (alpha) subunit of the CD8 co-receptor expressed on the surface of cytotoxic T cells and certain natural killer cells. The CD8 co-receptor is involved in the recognition of major histocompatibility complex (MHC) class I molecules, which play a crucial role in the adaptive immune response.

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Lab products found in correlation

Cd4 rm4 5, by Thermo Fisher Scientific (5 mentions) Cd62l mel 14, by BioLegend (3 mentions) Cd45 30 f11, by BioLegend (3 mentions) Cd44 im7, by Thermo Fisher Scientific (3 mentions) Tcrβ h57 597, by Thermo Fisher Scientific (3 mentions) Facsdiva software, by BD (2 mentions) Ack lysis buffer, by Thermo Fisher Scientific (2 mentions) Pd 1 j43, by Thermo Fisher Scientific (2 mentions) αcd16 32, by Cytek Biosciences (2 mentions) Lfa 1 clone h155 78, by BioLegend (2 mentions) Cd44 clone im7, by BD (2 mentions) Tim 3 rmt3 23, by BioLegend (2 mentions) Lag 3 c9b7w, by BioLegend (2 mentions) Cd19 1d3, by BD (2 mentions) Cd11c n418, by BD (2 mentions) Fortessa 1770 flow cytometer, by BD (2 mentions) Cell counting beads, by Merck Group (2 mentions) Klrg1 2f1, by BioLegend (2 mentions) Facsaria 2, by BD (2 mentions) Igd 11 26c 2a, by BioLegend (2 mentions)

Spelling variants of this product

CD8α (clone 53–6.7, (11 citations) Anti-CD8α (53–6.7, (6 citations) Anti-CD8α-PE (clone 53-6.7; (4 citations) Anti-CD8α APC (clone 53-6.7, (3 citations) Monoclonal antibodies to anti-CD8α (53–6.7, (2 citations) Anti-CD8α (53-6-7)-APC/eFluor780 (2 citations) Anti-CD8α-FITC (53-6.7; (2 citations) PE-Cy7 anti-CD8α (clone 53-6.7, (2 citations) MAbs to CD8α (clone 53-6.7; (2 citations) CD8α-APC-eFluor780 (clone 53-6.7) (2 citations)

17 protocols using cd8α 53 6

1

Multiparameter Flow Cytometry of PBMCs

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Peripheral blood mononuclear cells (PBMCs) were collected in 20 mM EDTA in Eppendorf tubes. PBMCs were spun for 8 minutes at 10,000 rpm at 20°C and serum was removed. RBCs were lysed with ACK Lysis buffer (GIBCO) for 8 minutes at room temperature (rt). If incomplete lysis occurred, cells were spun down for a second time at 12,000 rpm for 30 s and the ACK lysis step was repeated. Mononuclear cells were stained 1:100 with CB101–109/H-2Db-PE tetramer in the presence of Fc block 1:100 (αCD16/32, Tonbo), and monoclonal antibodies at 1:100 specific to CD45 (30-F11, Biolegend), CD8α (53–6.7, ebioscience), LFA-1 (clone H155–78, Biolegend), CD44 (clone IM7, BD), CD62L (MEL-14, Biolegend), PD-1 (J43, Invitrogen), KLRG1 (2F1, Biolegend), Tim-3 (RMT3–23, Biolegend), Lag-3 (C9B7W, Biolegend). CD19 (1D3, BD), F4/80 (T45–2342, BD), and CD11c (N418, BD) were used for a dump channel along with ghost dye BV510 at 1:500 (Tonbo). Antibodies were diluted in FACs buffer (PBS + 2.5% FBS) and cells were stained at room temperature in the dark for 60 minutes and then added to 100 μl of cell counting beads (Sigma Aldrich). Stained cells were fixed with 0.4% paraformaldehyde and stored overnight in the dark at 4°C prior to FACs acquisition using a Fortessa 1770 flow cytometer and Facs Diva software (BD Biosciences) and analyzed using FlowJo software (version 10).
Burrack A.L., Spartz E.J., Raynor J.F., Wang I., Olson M, & Stromnes I.M. (2019). Combination PD-1 and PD-L1 Blockade Promotes Durable Neoantigen-Specific T Cell-Mediated Immunity in Pancreatic Ductal Adenocarcinoma. Cell reports, 28(8), 2140-2155.e6.
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2

Murine T Cell Phenotyping by Flow Cytometry

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Single-cell suspensions isolated from indicated mouse organs were stained on ice with antibodies specific for CD4 (RM4–5; eBioscience), CD5 (53–7.3; eBioscience), CD8α (53–6.7; eBioscience), CD8β (YTS 156.7.7; Absolute Antibody), CD25 (3C7; BioLegend), CD44 (IM7; eBioscience), CD62L (MEL-14; BioLegend), CD69 (HI.2F3; BioLegend), CD122 (5H4; BioLegend), CD132 (TUGm2; BioLegend), and Vα2 (B20.1; BioLegend). For the staining with antibody specific for CCR7 (4B12; eBioscience), cells were incubated at 37 °C for 30 min before the staining with other antibodies. To isolate naïve CD8+ T cells, spleen cells were stained with biotinylated antibodies specific for CD4, CD44, and B220 (RA3–6B2; BioLegend), and cells that did not bind to the antibodies were enriched with magnetic bead conjugated streptavidin (Miltenyi Biotec). Multicolor flow cytometry and cell sorting were performed on FACSVerse and FACSAriaII (BD Biosciences), respectively.
Khanom U.S., Ohigashi I., Fujimori S., Kondo K., Takada K, & Takahama Y. (2019). TCR Affinity for In Vivo Peptide-Induced Thymic Positive Selection Fine-Tunes TCR Responsiveness of Peripheral CD8+ T Cells. The Journal of Immunology Author Choice, 203(4), 881-887.
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3

Single-cell Immunophenotyping and Glucose Uptake

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Single-cell suspensions were prepared and stained as previously described (Lin et al., 2016 (link)). Antibodies used include rabbit anti-myc tag (71D10, Cell Signaling Technology), mouse anti-myc tag (4A6, Millipore), TCF1 (C63D9, Cell Signaling Technology), Pax5 (1H9, eBioscience), IgM (Il/41, eBioscience), IgD (11-26c.2a, BioLegend), CD23 (B3B4, eBioscience), CD4 (RM405, BioLegend), CD8α (53-6.7, eBioscience), and Gr1 (RB6-8C5, BD PharMingen). Glucose uptake was determined by staining with the fluorescent glucose analog 2-NBDG (Cayman, 100 μM) in glucose-free RPMI.
Chen Y.H., Kratchmarov R., Lin W.H., Rothman N.J., Yen B., Adams W.C., Nish S.A., Rathmell J.C, & Reiner S.L. (2018). Asymmetric PI3K Activity in Lymphocytes Organized by a PI3K-Mediated Polarity Pathway. Cell reports, 22(4), 860-868.
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4

Isolation and Characterization of Murine Immune Cells

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Splenocytes were isolated as previously described(23 ). PBS-perfused kidneys and lungs were minced and digested with collagenase IV (100 μg/ml) for 30 minutes at 37 °C to prepare single cells.
Isolated cells were stained with the following antibodies specific for: TCRβ (H57-597, BioLegend), CD3ε (145-2C11, eBiosciences), CD4(GK1.5, BioLegend), CD8α (53-6.7, eBiosciences), B220 (RA3-6B2, BioLegend), CD25(PC61, BioLegend), I-A/I-E (M5/114.15.2, BioLegend), Nkp46 (29A1.4, BioLegend) for 30min at 4 °C. For intracellular staining, cells were stimulated with 20 ng/ml of phorbol-myristate acetate (PMA, Sigma) and 1 mg/ml of ionomycin(Sigma) for 4 hours, washed and stained with TCRβ, CD4, CD8, I-A/I-E. BD cytofix/cytoperm plus with Golgi stop staining kits (BD Biosciences) were used according to the manufacturer’s protocol. Antibodies of IFN-γ (XMG1.2, BioLegend) and IL-17A (TC11-18H10.1, BioLegend) were used for detecting intracellular cytokines.
Mizui M., Koga T., Lieberman L.A., Beltran J., Yoshida N., Johnson M.C., Tisch R, & Tsokos G.C. (2014). Interleukin-2 Protects Lupus-prone Mice from Multiple End-organ Damage by Limiting CD4−CD8− Interleukin-17-producing T-cells. Journal of immunology (Baltimore, Md. : 1950), 193(5), 2168-2177.
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5

Comprehensive Multicolor Flow Cytometry Protocol

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Extracellular staining was preceded by incubation with purified anti-CD16/32 antibodies (FcγRII/III block, 2.4G2) (eBioscience) to block non-specific staining. Cells were stained with FITC-, PE-, PerCP-Cy5.5-, PE-Cy5-, PE-Cy7-, APC-, and APC-H7- labelled appropriate antibodies including: TCRβ (H57-597, eBioscience); CD45 (30-F11, eBioscience and BD Biosciences); CD44 (IM7, eBioscience); CD8α (53–6.7, eBioscience); CD62L (MEL-14, eBioscience); CD4 (GK1.5, BD Biosciences and Miltenyi Biotec); or appropriate isotype Abs. Intranuclear FOXP3 staining was performed using eBioscience PE- or APC-conjugated FOXP3 staining buffer set (FJK-16 s). Intranuclear Ki-67 staining was performed using BD Pharmingen Ki-67 (B56) on permeabilized cells. Six-colour flow cytometry was performed with a FACSCanto cytometer (BD Biosciences). Data files were analysed using FlowJo software (Tree star Inc.).
Martinet K.Z., Bloquet S, & Bourgeois C. (2014). Ageing combines CD4 T cell lymphopenia in secondary lymphoid organs and T cell accumulation in gut associated lymphoid tissue. Immunity & Ageing : I & A, 11, 8.
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6

Immunological Antibody Staining for Flow Cytometry

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The following antibodies were used for immunofluorescence and immunohistochemistry: CD45R/B220 (RA3-6B2, BD Pharmingen), CD11c (N418, eBioscience), Fc receptor block;anti-CD16/CD32 (2.4G2, BD Pharmingen), CD3 (F7.2.38, Agilent Technologies), von Willebrand factor (vWF) (A0082, Dako). The following antibodies were used for flow cytometry: CD45.1 (A20, eBioscience), CD4 (RM4-5, eBioscience), IL-17A (TC11-18H10, BD Pharmingen), FoxP3 (FJK-16s, eBioscience), RoRγt (Q31-378, BD Pharmingen), CD44 (IM7, eBioscience), IFNγ (XMG1.2, BD Pharmingen), CD8α (53-6.7, eBioscience), γδ-TCR (eBioGL3, eBioscience). Anti-mCD4 (GK1.5, BioXCell) was used for in vivo CD4+ T-cell depletion.
Chung A.Y., Li Q., Blair S.J., De Jesus M., Dennis K.L., LeVea C., Yao J., Sun Y., Conway T.F., Virtuoso L.P., Battaglia N.G., Furtado S., Mathiowitz E., Mantis N.J., Khazaie K, & Egilmez N.K. (2014). Oral interleukin-10 alleviates polyposis via neutralization of pathogenic T-regulatory cells. Cancer research, 74(19), 5377-5385.
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7

Immunofluorescent Analysis of Splenic Immune Cells

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Spleens were frozen in Optimal Cutting Temperature (OCT) media (Sakura). Tissues were cut into 7-μm sections and treated with ice-cold acetone. Sections were stained with directly conjugated antibodies: CD8α (53–6.7, eBioscience), CD4 (RM4-5, eBioscience), and B220 (RA3-6B2, eBioscience). Nuclei were stained with 1 ug/mL DAPI. Stained slides were mounted with Prolong Gold antifade reagent (Life Technologies), imaged using a Nikon Eclipse 90i microscope and analyzed using Adobe Photoshop software.
Pestal K., Funk C.C., Snyder J.M., Price N.D., Treuting P.M, & Stetson D.B. (2015). Isoforms of the RNA editing enzyme ADAR1 independently control nucleic acid sensor MDA5-driven autoimmunity and multi-organ development. Immunity, 43(5), 933-944.
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8

Multiparametric Flow Cytometry of Immune Cells

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Monoclonal antibodies used were CD11c (HL3; BD), CD103 (2E7; BioLegend), CD11b (M1/70; eBioscience), CD64 (X54‐5/7.1; BioLegend), MHC class II (M5/114.15.2; eBioscience), CD45 (30‐F11; BioLegend), CD45R (RA3‐6B2; BD), CD8α (53‐6.7; eBioscience), CD197 (4B12; eBioscience), Plet1 (1D4), CD38 (90; eBioscience), CD19 (1D3; eBioscience), CD95 (Jo2; BD), Donkey anti‐rat IgG (Life Technologies), CD4 (GK1.5; BD), Foxp3 (Fjk‐16s; eBioscience), IL‐10 (JES5‐16E3; eBioscience), IFNγ (XMG1.2; BD), and IL‐17A (TC11‐18H10; BD Pharmingen). Zombie Aqua Fixable viability kit (BioLegend) was used for dead cell exclusion.
Karrich J.J., Romera‐Hernández M., Papazian N., Veenbergen S., Cornelissen F., Aparicio‐Domingo P., Stenhouse F.H., Peddie C.D., Hoogenboezem R.M., den Hollander C.W., Gaskell T., Medley T., Boon L., Blackburn C.C., Withers D.R., Samsom J.N, & Cupedo T. (2018). Expression of Plet1 controls interstitial migration of murine small intestinal dendritic cells. European Journal of Immunology, 49(2), 290-301.
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9

Multiparametric Flow Cytometry Analysis

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Cell suspensions were stained with antibodies for mouse studies as follows: FITC-conjugated B220 (RA3-6B2, eBioscience), CD3 (17A2, eBioscience), CD4 (RM4-5, eBioscience), CD5 (53–7.3, eBioscience), CD8α (53–6.7, eBioscience), CD11b (M1/70, eBioscience), CD11c (N418, eBioscience), CD19 (eBio1D3, eBioscience), Gr-1 (RB6-8C5, eBioscience), TCRβ (N57-597, eBioscience), Ter-119 (Ter119, eBioscience), γδTCR (eBioGL3, eBioscience), NK1.1 (PK136, eBioscience), FcεR1 (MAR-1, eBioscience). PE-conjugated ST2 (DJ8, MD Biosciences), PerCP/Cy5.5-cinjugated CXCR4 (Biolegend). APC-conjugated CD90.2 (53–2.1, eBioscience). Alexa Fluor 700-conjugated CD45 (30-F11, eBioscience). BV421-conjugated Ly-6A/E (D7, BD Biosciences). PE-Cyanine 7-conjugated KLRG1 (2F1, BD Biosciences).
To stain GRK2 antigen (R&D systems), cells were fixed and permeabilized after cell surface marker staining. Cells were analyzed by BD LSRII flow cytometer (BD Bioscences) and FlowJo software.
Lai D., Chen W., Zhang K., Scott M.J., Li Y., Billiar T.R., Wilson M.A, & Fan J. (2022). GRK2 regulates group 2 innate lymphoid cell mobilization in sepsis. Molecular Medicine, 28, 32.
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10

Multiparameter Flow Cytometry of PBMCs

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Peripheral blood mononuclear cells (PBMCs) were collected in 20 mM EDTA in Eppendorf tubes. PBMCs were spun for 8 minutes at 10,000 rpm at 20°C and serum was removed. RBCs were lysed with ACK Lysis buffer (GIBCO) for 8 minutes at room temperature (rt). If incomplete lysis occurred, cells were spun down for a second time at 12,000 rpm for 30 s and the ACK lysis step was repeated. Mononuclear cells were stained 1:100 with CB101–109/H-2Db-PE tetramer in the presence of Fc block 1:100 (αCD16/32, Tonbo), and monoclonal antibodies at 1:100 specific to CD45 (30-F11, Biolegend), CD8α (53–6.7, ebioscience), LFA-1 (clone H155–78, Biolegend), CD44 (clone IM7, BD), CD62L (MEL-14, Biolegend), PD-1 (J43, Invitrogen), KLRG1 (2F1, Biolegend), Tim-3 (RMT3–23, Biolegend), Lag-3 (C9B7W, Biolegend). CD19 (1D3, BD), F4/80 (T45–2342, BD), and CD11c (N418, BD) were used for a dump channel along with ghost dye BV510 at 1:500 (Tonbo). Antibodies were diluted in FACs buffer (PBS + 2.5% FBS) and cells were stained at room temperature in the dark for 60 minutes and then added to 100 μl of cell counting beads (Sigma Aldrich). Stained cells were fixed with 0.4% paraformaldehyde and stored overnight in the dark at 4°C prior to FACs acquisition using a Fortessa 1770 flow cytometer and Facs Diva software (BD Biosciences) and analyzed using FlowJo software (version 10).
Burrack A.L., Spartz E.J., Raynor J.F., Wang I., Olson M, & Stromnes I.M. (2019). Combination PD-1 and PD-L1 Blockade Promotes Durable Neoantigen-Specific T Cell-Mediated Immunity in Pancreatic Ductal Adenocarcinoma. Cell reports, 28(8), 2140-2155.e6.
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