Cd8a clone 53 6

Manufactured by BD

CD8a (clone 53-6.7) is a laboratory research reagent used for identification and enumeration of CD8+ T cells. It is a monoclonal antibody that binds to the CD8a molecule expressed on the surface of cytotoxic T lymphocytes. This reagent can be used in flow cytometry applications.

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Lab products found in correlation

Nk1.1 clone pk136, by BD (7 mentions) Cd4 clone rm4 5, by BD (5 mentions) Cd44 clone im7, by Thermo Fisher Scientific (5 mentions) Thy1.1 clone his51, by Thermo Fisher Scientific (5 mentions) Cd3 clone 145 2c11, by Thermo Fisher Scientific (5 mentions) Cd25 clone pc61, by BD (5 mentions) Cd11b clone m1 70, by BD (4 mentions) Gr1 clone rb6 8c5, by BD (3 mentions) Ter 119 clone ter119, by BD (2 mentions) Cd45r b220 clone ra3 6b2, by BD (2 mentions) Biotinylated goat anti rabbit igg, by Vector Laboratories (2 mentions) Lsrii flow cytometer, by BD (2 mentions) Cd4 clone gk1, by BD (1 mentions) Clone 30 f11, by BD (1 mentions) Facscanto flow cytometer, by BD (1 mentions) Sca 1 clone d7, by BD (1 mentions) F4 80 clone bm8, by BD (1 mentions) Cd11c clone n418, by BD (1 mentions) Facscalibur flow cytometer, by BD (1 mentions) Cellquest software, by BD (1 mentions)

17 protocols using cd8a clone 53 6

Immunophenotyping of Tumor-Infiltrating Leukocytes

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Transplanted melanoma infiltrating immune cells were isolated as described previously and stained with fluorochrome-conjugated monoclonal antibody specific for mouse CD45 (clone 30-F11, 1:200), CD11b (clone M1/70, 1:200), Gr1 (clone RB6–8C5, 1:200), CD8a (clone 53-6.7, 1:200) and CD4 (clone GK1.5, 1:200; all from BD Pharmingen) according to standard protocols. Data were acquired with a FACSCanto Flow Cytometer (BD Biosciences) and analysed with FlowJo software (TreeStar, V7.6.5 for Windows).

Riesenberg S., Groetchen A., Siddaway R., Bald T., Reinhardt J., Smorra D., Kohlmeyer J., Renn M., Phung B., Aymans P., Schmidt T., Hornung V., Davidson I., Goding C.R., Jönsson G., Landsberg J., Tüting T, & Hölzel M. (2015). MITF and c-Jun antagonism interconnects melanoma dedifferentiation with pro-inflammatory cytokine responsiveness and myeloid cell recruitment. Nature Communications, 6, 8755.

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Multiparametric Flow Cytometry Analysis

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Cells were stained with fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-, or allophycocyanin (APC)-conjugated monoclonal antibodies specific for mouse CD71 (clone C2), CD117 (clone 2B8), Sca-1 (clone D7), TER-119 (clone Ter-119), CD11b (clone M1/70), Gr-1 (clone RB6-8C5), F4/80 (clone BM8), CD11c (clone N418), CD317 (clone eBio927), CD4 (clone RM4-5), CD8a (clone 53-6.7), CD3e (clone 145-2C11), B220 (clone RA3-6B2), CD19 (clone 1D3), CD41 (clone MWReg30), CD42d (clone 1C2), NK-1.1 (clone PK136), and FceR1 (clone MAR-1) (from BD Biosciences and eBioscience). Stained cells were washed and analyzed by 4-color flow cytometry on a FACSCalibur flow cytometer (BD Biosciences) at the Flow and Image Cytometry Laboratory of University of Oklahoma Health Sciences Center. Data were collected by using the Cell Quest software (BD Biosciences) and analyzed by using the Summit software (Dako Colorado, Inc.). At least 15,000 total events were analyzed. Dead cells were excluded according to staining with 7-amino-actinomycin D. For apoptosis analysis, the cells were stained with FITC-Annexin V and propidium iodide.

Zhao W., Zou K., Farasyn T., Ho W.T, & Zhao Z.J. (2014). Generation and Characterization of a JAK2V617F-Containing Erythroleukemia Cell Line. PLoS ONE, 9(7), e99017.

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Skin Histology and Immunohistochemistry

Cited 1 time

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Dorsal skin was collected and processed as described previously for histology and immunohistochemistry^{44 (link)}. Formalin-fixed, paraffin-embedded skin was sectioned and stained with haematoxylin and eosin (H&E) using standard techniques as described^{44 (link)}. Fresh frozen skin was sectioned and stained, as previously published^{44 (link), 49 (link)}, using antibodies targeting the following proteins: CD4 (clone RM4-5, Cat.#550280), CD8a (clone 53-6.7, Cat.#550281), CD11c (clone HL3, Cat.#550283; all BD Pharmingen; San Jose, CA) and F4/80 (clone BM8, Cat.#14-4801, eBioscience; San Diego, CA).
Epidermal thickness (acanthosis) measurements and immune cell quantification in dorsal skin sections was completed using microscopic images collected from the stained sections using interactive image analyses approaches as described previously^{44 (link)}.

Arbiser J.L., Nowak R., Michaels K., Skabytska Y., Biedermann T., Lewis M.J., Bonner M.Y., Rao S., Gilbert L.C., Yusuf N., Karlsson I., Fritz Y, & Ward N.L. (2017). Evidence for biochemical barrier restoration: Topical solenopsin analogs improve inflammation and acanthosis in the KC-Tie2 mouse model of psoriasis. Scientific Reports, 7, 11198.

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Isolation and Characterization of Murine Hematopoietic Stem Cells

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Bone marrow cells were isolated from femurs, tibiae and iliac crests with PBS supplemented with 2% FCS and strained through 70 μm meshes. Red cells were lysed by resuspending the cells in 10 mL red cell lysis buffer (130-094-183, MACS Miltenyi Biotec) for 10 min at room temperature. After centrifugation, the cell pellet was resuspended in PBS supplemented with 2% FCS and nucleated cells were counted with 3% acetic acid on a Vi-Cell XR cell viability counter (Beckman Coulter). 10 × 10⁶ bone marrow cells were resuspended in 200 μL of PBS supplemented with 2% FCS containing the following antibody solution: FITC-conjugated lineage cocktail with antibodies against CD4 (clone H129.19, BD PharMingen), CD3e (clone 145-2C11, eBioscience), Ly-6G/Gr-1 (clone RB6-8C5, eBioscience), CD11b/Mac-1 (clone M1/70, BD PharMingen), CD45R/ B220 (clone RA3-6B2, BD PharMingen), FcεR1α (clone MAR-1, eBioscience), CD8a (clone 53-6.7, BD PharMingen), CD11c (clone N418, eBioscience), TER-119 (clone Ter119, BD PharMingen); c-Kit (PerCP-Cy5.5, clone 2B8, eBioscience), Sca-1 (PE-Cy7, clone D7, eBioscience), Flt3 (PE, clone A2F10, eBioscience), CD34 (eFluor660, clone RAM34, eBioscience), CD16/32 (BV421, clone 93, BioLegend) and Il-7R⍺ (BV605, clone A7R34, BioLegend).

Dingler F.A., Wang M., Mu A., Millington C.L., Oberbeck N., Watcham S., Pontel L.B., Kamimae-Lanning A.N., Langevin F., Nadler C., Cordell R.L., Monks P.S., Yu R., Wilson N.K., Hira A., Yoshida K., Mori M., Okamoto Y., Okuno Y., Muramatsu H., Shiraishi Y., Kobayashi M., Moriguchi T., Osumi T., Kato M., Miyano S., Ito E., Kojima S., Yabe H., Yabe M., Matsuo K., Ogawa S., Göttgens B., Hodskinson M.R., Takata M, & Patel K.J. (2020). Two Aldehyde Clearance Systems Are Essential to Prevent Lethal Formaldehyde Accumulation in Mice and Humans. Molecular Cell, 80(6), 996-1012.e9.

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Selective Expansion of Memory CD8+ T Cells

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Example 7

The potency of the IL-2 mutein H9 on the expansion of memory phenotype CD8⁺ T cells expressing low levels of CD25 but high levels of IL-2Rβγ was assessed in vivo. C57B1/6 mice received either PBS, 20 μg IL-2, 20 μg H9, or 1.5 μg IL-2/anti-IL-2 monoclonal antibody complexes and total cell counts of splenic CD3⁺ CD4⁺ CD44^highmemory phenotype T cells were assessed by flow cytometry. Splenic cell suspensions were prepared and stained with fluorochrome-conjugated monoclonal antibodies CD3 (clone 145-2C11, eBioscience), CD4 (clone RM4-5, Caltag Laboratories), CD8a (clone 53-6.7, BD Biosciences), CD25 (clone PC61, BD Biosciences), CD44 (clone IM7, eBioscience) NK1.1 (clone PK136, BD Biosciences) and Thy1.1 (clone HIS51, eBioscience). At least 100,000 viable cells were acquired using a BD FACSCanto™ II flow cytometer and analyzed using FlowJo software (TriStar, Inc.). As shown in FIG. 10A, treatment with the disclosed IL-2 mutein resulted in greater expansion of memory phenotype T cells relative to other treatment modalities with limited expansion of CD3⁺ CD4⁺ CD25^highT cells regulatory T cells (FIG. 10B).

US10654905B2. Method of treating graft versus host disease with an interleukin-2 mutein (2020-05-19). The Board of Trustees of the Leland Stanford Junior University [US], United States Department of Health and Human Services, Office of Technology Transfer, National Institutes of Health [US]. Inventors: Christopher K. Garcia [US], Suman Mitra [US], Warren J. Leonard [US], Aaron M. Ring [US].

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Memory CD8+ T Cell Expansion by IL-2 Mutein

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Example 7

The potency of the IL-2 mutein H9 on the expansion of memory phenotype CD8⁺ T cells expressing low levels of CD25 but high levels of IL-2Rβγ was assessed in vivo. C57B1/6 mice received either PBS, 20 μg IL-2, 20 μg H9, or 1.5 μg IL-2/anti-IL-2 monoclonal antibody complexes and total cell counts of splenic CD3⁺CD4⁺CD44^highmemory phenotype T cells were assessed by flow cytometry. Splenic cell suspensions were prepared and stained with fluorochrome-conjugated monoclonal antibodies CD8a (clone 53-2C11, eBioscience), CD4 (clone RM4-5, Caltag Laboratories), CD8a (clone 53-6.7, BD Biosciences), CD25 (clone PC61, BD Biosciences), CD44 (clone IM7, eBioscience) NK1.1 (clone PK136, BD Biosciences) and Thy1.1 (clone HIS51, eBioscience). At least 100,000 viable cells were acquired using a BD FACSCanto™ II flow cytometer and analyzed using FlowJo software (TriStar. Inc.). As shown in FIG. 10(A), treatment with the disclosed IL-2 mutein resulted in greater expansion of memory phenotype T cells relative to other treatment modalities with limited expansion of CD3⁺ CD4⁺ CD25^highT cells regulatory T cells FIG. 10(B).

US10183980B2. Superagonists and antagonists of interleukin-2 (2019-01-22). The Board of Trustees of the Leland Stanford Junior University, Office of the General Counsel [US]. Inventors: Kenan Christopher Garcia [US], Aron Levin [US], Aaron Ring [US].

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Immunohistochemistry and Flow Cytometry Protocol

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Five micron paraffin embedded tissue sections were processed for immuno-histochemistry as previously described (Sur et al., 2012b (link)). Rabbit polyclonal anti-Myc (Santa Cruz, sc-764; RRID:AB_631276) (1:500), Rabbit monoclonal anti Ki-67 (abcam, ab16667; RRID:AB_302459) (1:200), Goat polyclonal anti-Vimentin (Santa Cruz, sc-7557; RRID:AB_793998) (1:500), biotinylated goat anti-Rabbit IgG (Vector Laboratories, BA1000; RRID:AB_2313606) and biotinylated rabbit anti-Goat IgG (Vector Laboratories, BA5000; RRID:AB_2336126) (1:350) antibodies were used. For flow cytometry, single cell suspensions of spleen and bone-marrow and cells from peripheral blood were stained with Fc-block (CD16/CD32 clone 93, Biolegend, 101302, RRID:AB_312801) and subsequently with CD19 (clone 1D3, BD Biosciences, RRID:AB_11154223), TER119 (clone TER119, Biolegend 116210, RRID:AB_313711), CD3ε (clone 145–2 C11, Biolegend 100308, RRID:AB_312673), NK1.1(clone PK136, Biolegend, 108716, RRID:AB_493590), GR1/LY6G (clone RB6-8C5, Biolegend, 108410, RRID:AB_313375), CD4 (clone RM4-5, BD Biosciences, 563747) and CD8a (clone 53–6.7, BD Biosciences, 563332). Dead cells were visualized using Propidium iodide. Samples were analyzed using a BD LSRFortessa instrument.

Dave K., Sur I., Yan J., Zhang J., Kaasinen E., Zhong F., Blaas L., Li X., Kharazi S., Gustafsson C., De Paepe A., Månsson R, & Taipale J. (2017). Mice deficient of Myc super-enhancer region reveal differential control mechanism between normal and pathological growth. eLife, 6, e23382.

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Multi-panel Flow Cytometry Immunophenotyping

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Flow cytometry experiments were performed on Aurora (Cytek Biosciences), BD Symphony 3 Flow cytometer, BD FACS ARIA III Sorter, and BD Melody Cell Sorter from the flow core in the University of Pittsburgh or Center for Discovery and Innovation and analyzed by Flowjo (BD). CD45 (clone 30-F11), CD4 (clone RMT4-5), CD8a (clone 53.67), Tcf1/Tcf7 (clone C63D9), PD-1 (clone J43), Tim-3 (clone RMT3-23), Lag-3 (clone C9B7W), Thy1.2 (clone 53-2.1), IFN-γ (clone XMG1.2), Ly-6C (clone HK1.4), Ly-6G (clone 1A8), Sca-1 (clone D7), ST2 (clone RMST2-33), CD103 (clone M290), GzmB (clone QA16A02), Foxp3 (clone MF-14), Ki-67 (clone 16A8), CD11b (clone M1/70), CD11c (clone N418), CD86 (clone GL-1), MHCII (clone), F4/80 (clone BM8), CD206 (clone C068C2), Arg1 (clone A1exF5), CD25 (clone PC61), CD62L (clone MEL-14), CD44 (clone IM7), CD90.2 (clone 53-2.1), CD140a (clone APA5), LIVE/DEAD dye (Zombie NIR Dye) were purchased from BD Bioscience, Thermo Fisher Scientific, or BioLegend. For intracellular transcription factors and cytokines staining, cells were stimulated with a leukocyte activation cocktail (BD) for 6 hours and then followed the standard staining protocol described previously (49 ).

Sun R., Zhao H., Gao D.S., Ni A., Li H., Chen L., Lu X., Chen K, & Lu B. (2023). Amphiregulin couples IL1RL1+ regulatory T cells and cancer-associated fibroblasts to impede antitumor immunity. Science Advances, 9(34), eadd7399.

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Multiparametric Flow Cytometry Analysis of Lung Cells

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The following directly conjugated antibodies were utilized for flow cytometry analysis: CD3 (clone 145-2C11; eBioscience), CD4 (clone GK1.5; Biolegend), CD8a (clone 53-6.7; BD Biosciences), CD11b (clone M1/70; Biolegend), CD11c (clone N418; Biolegend), CD19 (clone 6D5; Biolegend), F4/80 (clone BM8; Biolegend), and GR-1 (Ly-6G) (clone RB6-8C5; eBioscience). All antibodies were titrated on normal B6 splenocytes prior to use. Lung cells were prepared as above. After preparation of single cell suspensions, cells were blocked for 30 min at 4°C with 24G2 serum to block Fc receptors. Cells were then stained with indicated antibodies for 30 min at 4°C in the dark. Cells were then washed three times with PBS + 2%BSA and analyzed on a Becton Dickinson LSRII flow cytometer. FlowJo (Treestar) was used for all flow cytometry analysis.

Powell D.A, & Frelinger J.A. (2017). Efficacy of Resistance to Francisella Imparted by ITY/NRAMP/SLC11A1 Depends on Route of Infection. Frontiers in Immunology, 8, 206.

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Lung Leukocyte Profiling in Mice

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In mouse models, BAL was performed by cannulation through the trachea and lavage with 1.5 ml sterile PBS. Cells were pelleted by centrifugation, resuspended in ACK buffer to lyse red cells, washed in PBS and resuspended in RPMI medium with 10% FBS. Cells were stained with Quik Diff (Reagena) for differential counting. For flow cytometry analysis, lung leukocytes were obtained from BAL and red cells were lysed with ACK buffer. BAL cells were stained with Live/Dead fixable dead cell stain (Life Technologies, Carlsbad, CA), incubated with anti-mouse CD16/CD32 (FC block; BD Biosciences) and subsequently with directly fluorochrome-conjugated monoclonal antibodies specific for CD3ε (clone 500 A; 2 1 µg/ml, BD Biosciences), CD69 (clone H1.2F3 1 µg/ml, BD Biosciences), CD4 (clone RM4-5 0.25 µg/ml; BD Biosciences), CD8a (clone 53–6.7; 0.5 µg/ml BD Biosciences) and NK1.1 (clone PK136;1 µg/ml, BD Biosciences). Data were acquired on an LSR II flow cytometer (BD Biosciences) and analysed using FlowJo software (version 10.0.6; Tree Star, Ashland, USA). Representative gating strategies used for analysis of cell surface staining are shown in Supplementary Fig. 8.

Singanayagam A., Glanville N., Girkin J.L., Ching Y.M., Marcellini A., Porter J.D., Toussaint M., Walton R.P., Finney L.J., Aniscenko J., Zhu J., Trujillo-Torralbo M.B., Calderazzo M.A., Grainge C., Loo S.L., Veerati P.C., Pathinayake P.S., Nichol K.S., Reid A.T., James P.L., Solari R., Wark P.A., Knight D.A., Moffatt M.F., Cookson W.O., Edwards M.R., Mallia P., Bartlett N.W, & Johnston S.L. (2018). Corticosteroid suppression of antiviral immunity increases bacterial loads and mucus production in COPD exacerbations. Nature Communications, 9, 2229.

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