Cd8 clone 53 6

Manufactured by Thermo Fisher Scientific

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The CD8 (clone 53-6.7) is a lab equipment product offered by Thermo Fisher Scientific. It is used for the detection and enumeration of CD8-positive cells in flow cytometric analysis.

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CD8 (53-6.7, (27 citations) Anti-CD8 (clone 53-6.7, (18 citations) Clone 53-6.7 (10 citations) CD8-PerCP-Cy5.5 (clone 53-6.7, (5 citations) Anti-CD8 (Clone 53–6.7) antibodies (2 citations) Anti-CD8-APC clone 53-6.7 (2 citations) Anti-mouse CD8 (clone 53–6.7, (2 citations) Anti-CD8 Cy5 (clone 53-6.7, (2 citations) Anti-CD8 clone 53-6.7 Armenian hamster IgG (2 citations) E-flour 615 CD8 (clone 53-6.7; (2 citations)

20 protocols using cd8 clone 53 6

Characterization of Tumor-Infiltrating Lymphocytes

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Single cell suspensions from spleens and lymph nodes were prepared as described.6 (link) TIL were isolated from pooled tumors as described.26 (link) All flow cytometry experiments were performed at least 3 times. Single cell suspensions were incubated with mouse Fc receptor binding inhibitor for 10 minutes before staining with antibodies to CD45 (clone 30-F11), CD3 (clone 145-2C11), CD4 (clone GK1.5), CD8 (clone 53-6.7), CD19 (clone eBio1D3), CD86 (clone GL1), CD11b (clone M1/70), Gr-1 (clone RB6-8C5), CD69 (clone H1.2F3), CD44 (clone IM9), CD62L (clone MEL-14), and CD11c (clone N418; all from eBioscience, San Diego, CA) for 30 minutes. Flow cytometry was performed using FACS Calibur (BD Biosciences) and the lymphocyte population was selected by gating CD45⁺ cells. The data were analyzed using Flow Jo software (Tree Star, Ashland, OR). For tetramer staining, 10 μL of PE labeled HLA-A*02:01 Human HPV16 E7 tetramer (NIH Tetramer Core Facility) was added to 200 μL mouse lymphocyte suspension (1×106 cells per tube). After incubation for 30 minutes, cells were centrifuged and resuspended in phosphate-buffered saline with 1% paraformaldehyde and then analyzed by flow cytometry. PE labeled HLA-A*02:01 human mesothelin tetramer (NIH Tetramer Core Facility) was used as control.

Dai M., Hellstrom I., Yip Y.Y., Sjögren H.O, & Hellstrom K.E. (2018). Tumor Regression and Cure Depends on Sustained Th1 Responses. Journal of Immunotherapy (Hagerstown, Md. : 1997), 41(8), 369-378.

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Multiparameter Flow Cytometry Immunophenotyping

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CD45R/B220 (clone RA3-6B2, 1:100, eBioscience), CD3 (145-2c11, 1:100, eBioscience), CD45.2 (clone 104, 1:100, eBioscience), CD4 (clone GK1.5, 1:100, eBioscience), CD8 (clone 53-6.7, 1:100, eBioscience), CD19 (clone eBio1D3, 1:100, eBioscience), CD5 (clone 53-7.3, 1:100, eBioscience), CD21 (clone eBio4E3, 1:100, eBioscience), CD43 (clone eBioR2/60, 1:100, eBioscience), IgD (clone 11–26, 1:100, eBioscience), Gr1 (clone RB6-8C5, 1:100, eBioscience), IgM (clone 11f41, 1:100, eBioscience), GL-7 (clone GL-7, 1:100, eBioscience), Mac1 (clone M1/70, 1:100, eBioscience).

Souroullas G.P., Fedoriw Y., Staudt L.M, & Sharpless N.E. (2017). Lkb1 deletion in murine B lymphocytes promotes cell death and cancer. Experimental hematology, 51, 63-70.e1.

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Phenotypic Profiling of Tumor-Infiltrating Cells

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Harvested tumours were collected in HBSS medium, minced and incubated 30 min at 37°C in non‐enzymatic cell dissociation buffer followed by mechanical dissociation through a 70 µm filter. Viable cells were then enriched using Ficoll gradient. All cell suspensions were counted, and one million viable cells were seeded in 96‐well plates in 100 µL of staining buffer for acquisition. Non‐specific binding was performed using mouse FcR blocking reagent. Fixable Viability Dye eFluor 780 (eBiosciences, 65‐0865‐14) was used to assess cell viability. Antibodies directed against the CD45 (clone 30‐F11, Biolegend, 103149), CD3 (clone 17A2, BD, 740268), CD8 (clone 53−6.7, eBiosciences, 61‐0081‐82), and anti‐PD‐L1 (clone 10F.9G2, Biolegend, 124334) were added. Stained cells were analyzed with a Fortessa X20 cytometer (BD Biosciences).

Jurisic A., Sung P., Wappett M., Daubriac J., Lobb I.T., Kung W., Crawford N., Page N., Cassidy E., Feutren‐Burton S., Rountree J.S., Helm M.D., O'Dowd C.R., Kennedy R.D., Gavory G., Cranston A.N., Longley D.B., Jacq X, & Harrison T. (2024). USP7 inhibitors suppress tumour neoangiogenesis and promote synergy with immune checkpoint inhibitors by downregulating fibroblast VEGF. Clinical and Translational Medicine, 14(4), e1648.

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Comprehensive Immunophenotyping of T Cells

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The cells were stained with the following antibodies: CD8 (clone 53–6.7, eBioscience), CD4 (clone GK1.5, eBioscience), CD90.1 (clone OX-7, eBioscience), CD90.2 (clone 53–2.1, eBioscience), PD-1 (clone J43, Biolegend), KLRG1 (clone 2F1, Biolegend), p53 (clone pAb 240, Novus Biologicals) TNF (Clone MP6-XT22, Biolegend) and IFNγ (clone XMG1.2, Biolegend) at the appropriate dilution and with compatible fluorochromes. The lymphocyte gates in the samples were plotted directly to examine CD8 T cell populations depicted in the flow plots.

Kurup S.P., Moioffer S.J., Pewe L.L, & Harty J.T. (2020). P53 hinders CRISPR/Cas9 mediated targeted gene disruption in memory CD8 T cells in vivo. Journal of immunology (Baltimore, Md. : 1950), 205(8), 2222-2230.

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Isolation and characterization of splenic DC subsets

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Dendritic cells were harvested from the spleen of mice 18–24 hours post immunization with 50μg polyI:C and 50μg αCD40 given intraperitoneally in phosphate buffered saline (PBS) as described in [19 (link)]. Briefly, spleens were macerated using forceps and digested at 37 degrees Celsius (C) with collagenase and DNAse (Worthington, Lakewood, NJ) in EHAA media without L-glutamine (GIBCO, Grand Island, NY) for 45 minutes. The digestion was stopped with 0.1M EDTA in Hank’s buffered saline solution and incubated for 5min at 37 degrees C. Single cell suspensions were made by pushing residual tissue pieces through a screen and washed with 5mM EDTA in EHAA. Following ACK (Ammonium-Chloride-Potassium) lysis cells were washed and stained with CD11c clone N418, CD8 clone 53–6.7, CD11b clone M1/70 (eBioscience, San Diego, CA), CD27 clone Lg.3A10 and CD70 clone FR70. All antibodies were purchase from Biolegend (San Diego, CA) unless otherwise stated. Cells were run on the DakoCytomation CyAn ADP flow cytometer (Fort Collins, CO) and acquired using Summit acquisition software. Gating strategy for the identification of CD8+ and CD11b+ splenic DC subsets, as well as for the identification of CD8+ T cells, is shown in supplemental Fig 3. FlowJo software (Tree Star, Ashland, OR) was used to analyze flow cytometry data and cells were counted using a ViCell (Beckman, Coulter, Brea, CA).

Burchill M.A., Tamburini B.A, & Kedl R.M. (2015). T cells compete by cleaving cell surface CD27 and blocking access to CD70 bearing APCs. European journal of immunology, 45(11), 3140-3149.

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Characterization of LCMV-specific CD8+ T cells

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The following mAb were purchased from eBioscience or Biolegend and used in an appropriate combination of fluorochromes: CD8 (clone 53-6.7; eBioscience), PD-1 (clone J43; eBioscience), LAG-3 (clone eBioC9B7W; eBioscience), 2B4 (clone eBio244F4; eBioscience), CD11a (clone M17/4; eBioscience), Thy1.1 (clone HIS51; eBioscience), IFNγ (clone XMG1.2; Biolegend) and TNFα (clone MP6-XT22; Biolegend). All LCMV-specific peptides were synthesized by Bio-Synthesis Inc (Bio-Synthesis, Louisville, TX): GP276-286 (SGVENPGGYCL) and GP33-41 (KAVYNFATM) (52 (link)). p:MHC class I tetramer H-2D^b GP276 and GP33 were made and used as previously described (25 (link), 48 (link)).

Condotta S.A., Khan S.H., Rai D., Griffith T.S, & Badovinac V.P. (2015). Poly-microbial sepsis increases susceptibility to chronic viral infection and exacerbates CD8+ T cell exhaustion. Journal of immunology (Baltimore, Md. : 1950), 195(1), 116-125.

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Identification of Adoptively Transferred T Cells

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Cell suspensions prepared from grafts or renal LN were incubated in round-bottom 96-well plates in 200 μl RPMI containing 10% FCS plus 20 ng ml⁻¹ PMA and 1 μg ml⁻¹ Ionomycin at 37 °C/5% CO₂. After 1 h BD GolgiStop (0.7 μl ml⁻¹) was added, then cells were cultured for an additional 3 h. The cell surface was stained for expression of CD4 (clone RM4-5, 1:400), CD8 (clone 53-6.7, 1:200), CD45.1 (clone A20⁻, eBioscience, 1:100) and CD45.2 (Clone 104, BioLegend, 1:150) enabling identification of host cells (CD4⁺CD8⁻CD45.2⁺ or CD4⁻CD8⁺CD45.2⁺) and adoptively transferred OT-I cells (CD4⁻CD8⁺CD45.1⁺). Cells were then fixed and permeabilized using BD Cytofix/Cytoperm reagents before staining for IL-2 expression (clone JES6-5H4, 1:200) and analysis by flow cytometry.

Sutherland R.M., Londrigan S.L., Brady J.L., Carrington E.M., Marchingo J.M., Heinzel S., Hodgkin P.D., Graham K.L., Kay T.W., Zhan Y, & Lew A.M. (2017). Cognate antigen engagement on parenchymal cells stimulates CD8+ T cell proliferation in situ. Nature Communications, 8, 14809.

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Immunohistochemical Staining of Skin Sections

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Immunohistochemical staining of formalin-fixed, paraffin-embedded skin sections was carried out using anti-CD4 (clone RM4-5, eBioscience), CD8 (clone 53-6.7, eBioscience) and MHC I (clone H100-5-28, antibodies-online.com) antibodies as previously described [11 (link)]. Biotinylated goat anti-rat/mouse IgGs (Abcam) were used as secondary antibody. Streptavidin-HRP staining was assayed using a standard DAB histochemistry kit (Abcam).

Gao Z., Jin Y.Q, & Wu W. (2017). SOCS3 treatment prevents the development of alopecia areata by inhibiting CD8+ T cell-mediated autoimmune destruction. Oncotarget, 8(20), 33432-33443.

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Cytokine profiling of obese WT and IFNAR-/- mice

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eWAT and liver was isolated from obese WT and IFNAR^−/− mice. eWAT-isolated SVF cells and liver immune cells were stimulated for 4 h with PMA (50 ng/ml; Sigma-Aldrich) and Ionomycin (1 μg/ml; Calbiochem). Briefly, cells were stained with Live/dead stain (Zombie UV Dye; 1:250; Biolegend), B220 (clone RA3-6B2; 1:100; Biolegend), CD45 (clone 104; 1:500), CD11b (clone M1/70; 1:100), F4/80 (clone BM8; 1:100), Gr1 (clone RB6-8C5; 1:100), CD4 (clone GK1.5; 1:50), CD8 (clone 53-6.7; 1:100), IFNγ (clone XMG1.2; 1:100) TNF (clone MP6-XT22; 1:100), and IL-6 clone (MP5-20F3; 1:100) (all ebioscience). Data were collected using a LSR Fortessa flow cytometer (BD Biosciences) and analyzed by FlowJo software (Tree Star).

Chan C.C., Damen M.S., Moreno-Fernandez M.E., Stankiewicz T.E., Cappelletti M., Alarcon P.C., Oates J.R., Doll J.R., Mukherjee R., Chen X., Karns R., Weirauch M.T., Helmrath M.A., Inge T.H, & Divanovic S. (2020). Type I interferon sensing unlocks dormant adipocyte inflammatory potential. Nature Communications, 11, 2745.

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Isolation and Activation of T Cell Subsets

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Single cell suspensions obtained from isolated lymph nodes and spleens were subjected to surface staining using the according antibodies (CD4: clone RM4-5, BioLegend; CD62L: clone MEL-14, eBiosciences; CD25: clone PC61.5, BioLegend; CD8: clone 53-6.7, eBiosciences). Cell sorting was carried out on the BD FACS Aria II (BD Biosciences) and purity of isolated cell subsets was generally >97%. For activation and expansion, cells were placed in cell culture dishes (Nunc) which had been coated with 1 µg/ml anti-CD3 (clone 145-2C11, eBiosciences) and 1 µg/ml anti-CD28 (clone 37.51, eBiosciences) in PBS over night at 4°C. Cells were seeded in supplemented RPMI 1640 L-Glutamine medium containing 10 ng/ml (Tconv) or 50 ng/ml (Tregs) IL-2 (R&D Systems).

Schreiber L., Pietzsch B., Floess S., Farah C., Jänsch L., Schmitz I, & Huehn J. (2014). The Treg-Specific Demethylated Region Stabilizes Foxp3 Expression Independently of NF-κB Signaling. PLoS ONE, 9(2), e88318.

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