Cells were harvested and stained from the thymus and other organs. Data were acquired on LSR Fortessa or LSRII flow cytometers (BD Biosciences) and analyzed using software designed by the Division of Computer Research and Technology, NCI. Live cells were gated using forward scatter exclusion of dead cells stained with propidium iodide. The following antibodies were used for staining: TCRβ (H57–597), CD24 (30-F1), IL-7Rα (A7R34), NK1.1 (PK136), IL-2Rβ (TM-β1), IL-4Rα (M1), RORγt (Q31–378), T-bet (4B10), PLZF (9E12), and isotype control antibodies, all from eBioscience; CD44 (IM7), γc (4G3), CD4 (GK1.5 and RM4.5), and CD8α (53–6-7) from BD Biosciences; IL-21R (4A9) and CD45 (30-F11) from BioLegend. CD1d tetramers loaded with PBS-57 and unloaded controls were obtained from the NIH tetramer facility (Emory University, Atlanta, GA). Active caspase-3 was determined using the CaspGLOW™ fluorescein active caspase-3 staining kit (eBioscience).
Cd8α 53 6.7
Manufactured by BD
Sourced in United States
CD8α (53-6.7) is a monoclonal antibody that recognizes the alpha chain of the CD8 glycoprotein. CD8 is a cell surface receptor that is expressed on a subset of T cells, known as cytotoxic T cells, and on a subset of natural killer cells. The CD8α (53-6.7) antibody can be used to identify and study these cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Caspglow fluorescein active caspase 3 staining kit, by Thermo Fisher Scientific (2 mentions)
Il 21r 4a9, by BioLegend (2 mentions)
Cd45 30 f11, by BioLegend (2 mentions)
Lsrfortessa, by BD (2 mentions)
Facscanto 2, by BD (2 mentions)
Cd4 rm4 5, by BD (2 mentions)
Streptavidin, by BD (1 mentions)
Peanut agglutinin, by Vector Laboratories (1 mentions)
Cd3 17a2, by BD (1 mentions)
Tcrγδ gl3, by BioLegend (1 mentions)
Maackia amurensis lectin 2, by Vector Laboratories (1 mentions)
Fortessa x20, by BD (1 mentions)
Sambucus nigra lectin, by Vector Laboratories (1 mentions)
Testosterone propionate, by Merck Group (1 mentions)
Ulex europaeus agglutinin 1 uea 1, by Vector Laboratories (1 mentions)
Anti cd4 rm4 5, by Thermo Fisher Scientific (1 mentions)
B220 ra3 6b2, by Thermo Fisher Scientific (1 mentions)
I a 1 e m5 114, by BioLegend (1 mentions)
Foxp3 staining kit, by Thermo Fisher Scientific (1 mentions)
Gr 1 rb6 8c5, by Thermo Fisher Scientific (1 mentions)
12 protocols using cd8α 53 6.7
1
Immunophenotyping of Thymic Subsets
2
Multiparameter Flow Cytometry Analysis of Immune Cell Populations
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Organs were collected from WT, CD45.1, CD45.2, OT-I, or CD8β-deficient mice and single-cell suspensions prepared using standard protocols. Following removal of red blood cells using ACK, nonspecific receptors were blocked with monoclonal antibody (mAb) 2.4G2, before cells (1–5 × 106) were stained with mAb to mouse NKp46 (29A1.4; BioLegend), CD3 (17A2; BD Biosciences), TCRγδ (GL3, BioLegend), TCRαβ (H57-597, BioLegend), CD8α (53-6.7; BD Biosciences), CD8β (H35-17.2; BD Biosciences). Alternatively cells were stained with the lectins peanut agglutinin (Vector Laboratories), sambucus nigra lectin (Vector Laboratories), or maackia amurensis lectin II (Vector Laboratories) before detecting using Streptavidin (BD Biosciences). Cells stained with tetramers were fixed in 2% paraformaldehyde for 15 min and washed twice with FACS buffer (1% FCS/PBS) before being resuspended in FACS buffer. All other FACS combinations were acquired unfixed. For acquisition, events were electronically gated on FSC-A versus FSC-H (singlets), followed by FSC-A and SSC-A (to exclude doublets and debris). Among the remaining population, at least 5000 electronic events of interest were collected using an LSR-II or X- 20 Fortessa (BD Biosciences).
3
Modulating Androgen Levels in Mice
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Degarelix (as acetate), a third generation LHRH-Ant (Firmagon), was resuspended in sterile water for injection and administered s.c. to mice at a dose of 40 µg/g. Lupron (11.25-mg 3-mo depot), an LHRH-Ag, was prepared according to the manufacturer’s instructions and administrated intramuscularly to mice at a dose of 20 µg/g. Degarelix and Lupron were purchased from the MSKCC Pharmacy. Testosterone propionate (Sigma-Aldrich) was resuspended in peanut oil and injected daily s.c. (1 mg/mouse) in 100 µl. Surface antibodies against CD44 (IM7), EpCAM (G8.8), PDGFRa (APA5), PECAM-1 (390), CD45 (30-F11), and H-2Kk (AF3-12.1.3) were purchased from eBioscience; anti-Ly-51 (BP-1), CD34 (RAM34), CD62L (MEL-14), H-2Kb (AF6-88.5), IFN-γ (XMG1.2), IL-2 (JES6-5H4), c-Kit (2B8), CD3ε (145-2C11), CD25 (PC61), TER-119 (TER-110), and CD8α (53–6.7) were purchased from BD; anti-CD4 (RM4-5) and B220 (RA3-6B2) were purchased form Invitrogen; anti-CD44 (IM7), CD90.2 (30-H12), and IA/IE (M5/114.15.2) were purchased from BioLegend; and Ulex europaeus agglutinin 1 (UEA-1) was purchased from Vector Laboratories. For in vitro cultures of T cell differentiation (Fig. 2, A and B ), cells were stained with antibodies to lineage (Lin)-specific markers: CD8, CD11c, NK1.1, CD3, CD4, B220, CD11b, and GR-1.
4
Comprehensive Immune Cell Analysis
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Single cell suspensions were prepared from the thymus of indicated mice. Data were acquired using LSR Fortessa or LSRII flow cytometers (BD Biosciences) and analyzed using FlowJo. Live cells were gated by forward scatter exclusion of dead cells stained with propidium iodide. The following antibodies were used for staining: TCRβ (H57–597), HSA (30-F1), IL-7Rα (A7R34), NK1.1 (PK136), IL-2Rα (PC61.5), IL-2Rβ (TM-β1), IL-4Rα (M1), Foxp3 (FJK-16s), RORγt (AKFJS-9) and isotype control antibodies, all from eBioscience; TCRγδ (GL3), CD44 (IM7), γc (4G3), CD4 (GK1.5 and RM4.5), and CD8α (53-6-7) from BD Biosciences; IL-9Rα (RM9A4), Bcl-2 (BCL/10C4), PLZF (9E12), and IL-21R (4A9) from BioLegend. Fluorochome-conjugated CD1d tetramers loaded with PBS-567 and unloaded controls were obtained from the NIH tetramer facility (Emory University, Atlanta, GA). Intranuclear Foxp3, PLZF, and RORγt proteins were detected using a Foxp3 staining kit according to the manufacturer’s instructions (eBioscience). Active caspase-3 induction was determined using the CaspGLOW™ fluorescein active caspase-3 staining kit (eBioscience).
5
Isolation of Murine Hematopoietic Stem Cells
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Single-cell suspensions of whole BM cells were labeled by incubation on ice for 15 min using a cocktail of the following antibodies: PE-conjugated anti-mouse CD3ε (145-2C11; BD Biosciences), CD4 (L3T4; BD Biosciences), CD8α (53-6.7; BD Biosciences), B220 (RA3-6B2; BD Biosciences), Mac-1 (M1/70; BD Biosciences), Gr-1 (RB6-8C5; eBioscience), and Ter119 (TER-119, eBioscience); FITC-conjugated anti-mouse Sca-1 (E13-161.7; BD Biosciences); and APC-conjugated anti-mouse c-Kit (2B8; BD Biosciences). After cells were washed with PBS, they were incubated with anti-PE microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) at 4°C for 15 min. Lin+ cells (CD4+, CD8α+, B220+, Mac-1+, Gr-1+, Ter119+ cells) were removed by immunomagnetic selection using a MACS system (Miltenyi Biotec). These negatively selected cells were subjected to FACS using a Moflo XDP cell sorter (Beckman-Coulter) to prepare LSK cells.
6
Multicolor Flow Cytometry of Immune Cells
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Membrane and intracellular staining of MLN or epithelial cells were performed as described.22 The following antibodies were used: CD4 (GK1.5, BioLegend), CD8α (53–6.7, BD Biosciences), CD16/CD32 (93, BioLegend), IL-17A (TC11-18H10.1, BioLegend), RORγt (Q31-378, BD Biosciences), IFNγ (XMG1.2, BD Biosciences), IL-10RA (1B1.3a, BioLegend), and Ep-CAM (G8.8, BioLegend).
7
Multiparametric Flow Cytometry Analysis
8
Characterization of Myeloid Cells in Pancreatic Tumors
9
Multiparametric Flow Cytometry Analysis of Immune Cells
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The BALF cells were stained with APC-anti-CD3 (145-2C11; BD Biosciences; T cells), APC-anti-B220 (RA3-6B2; eBioscience; B cells), FITC-anti-I-A/I-E (clone M5/114.15.2; eBioscience; DCs/macrophages), PE-Cy7-anti-CD11c (N418; eBioscience), PE-anti-CCR3 (83101; R&D Systems; eosinophils) (23 (link)). For splenic DC subset analysis, splenocytes were stained with APC-anti-CD11c (N418; eBioscience), PE-anti-PDCA-1 (129c; eBioscience), CD4 (RM4-5; BD Biosciences), CD8α (53-6.7; BD Biosciences), or anti-IGF2R polyclonal Ab (R&D). Absolute number of each DC subset was calculated from total splenocytes. For dead cell detection, purified splenic cDCs were treated with ODN 1826 (10 µg/ml, InvivoGen) or anti-mouse CD40 mAb (20 µg/ml, BD Biosciences) in the presence of human Leu27-IGF2 (Novozymes GroPep, Thebarton, Australia) for 24 hours, and then stained with flourchrome-conjugated antibodies against CD11c, CD4, and CD8α and FITC-Annexin V and ViViD (Invitrogen). The cellular composition of BALF cells and splenic DC subsets were analyzed with a multi-parametric flow cytometer (LSR II; BD Biosciences) and FlowJo software (version 10, Tree Star, Inc, Ashland, OR).
10
Tumor Immune Cell Analysis in MMTV-PyMT Mice
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The MMTV-PyMT model was prepared as described above. Mice were treated on day 7 with aldoxorubicin, Dox-SA, or Dox-CBD-SA (5 mg/kg). Mice were euthanized on day 14. Cell suspensions were obtained from each tumor as described previously (9 (link)). Tumors were harvested and digested in DMEM supplemented with 2% FBS, collagenase D (2 mg/ml), and DNase I (40 μg/ml) (Roche) for 30 min at 37°C. Single-cell suspensions were obtained by gently disrupting the organs through a 70-μm cell strainer. Red blood cells were lysed with ACK lysing buffer (Quality Biological). Fixable live/dead cell discrimination was performed using Fixable Viability Dye eFluor 455 (eBioscience) according to the manufacturer’s instructions. Following a washing step, cells were stained with specific antibodies for 20 min on ice before fixation. The following antibodies were used to stain the cells: CD3 (145-2C11, BD Biosciences), CD4 (RM4-5, BD Biosciences), CD8α (53-6.7, BD Biosciences), CD45 (30-F11, BD Biosciences), and NK1.1 (PK136, BD Biosciences). All flow cytometric analyses were done using a Fortessa flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star).
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