Single-cell suspensions from spinal cords were obtained via mechanical dissociation on a cell strainer. Immune cells were separated over a two-phase Percoll-density gradient. Staining of αβTCR/CD4+ T cells, αβTCR/CD8+ T cells and CD45/CD11b cells (macrophages/microglia) was performed using the following antibodies in a 1:200 dilution: Anti-CD3e (clone 145-2C11), BioLegend; anti-CD4 (clone GK 1.5), BD; anti-CD8 (clone 53-6.7), BD; anti-CD8 (clone 53–6.7), BD; anti-CD11b (clone M1/70), BioLegend; anti-CD45.2 (clone 104), BioLegend. The addition of Calibrite APC beads (BD) allowed for cell quantification. Flow cytometry was performed using a FACSCalibur operated by Cell Quest software (Becton Dickinson).
Anti cd8 clone 53 6
Manufactured by BD
Sourced in United States
Anti-CD8 (clone 53-6.7) is a mouse monoclonal antibody that recognizes the CD8 cell surface glycoprotein. CD8 is a co-receptor expressed on the surface of cytotoxic T cells and a subset of natural killer cells. The antibody can be used to identify and enumerate CD8+ cells in flow cytometry applications.
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Lab products found in correlation
Anti cd4 clone gk1.5, by BD (5 mentions)
Anti cd3e clone 145 2c11, by BioLegend (3 mentions)
Calibrite apc beads, by BD (3 mentions)
Anti cd45.2 clone 104, by BioLegend (3 mentions)
Anti cd11b clone m1 70, by BioLegend (3 mentions)
Anti cd11b clone m1 70, by BD (3 mentions)
Anti cd4 clone rm4 5, by BD (3 mentions)
Facscalibur, by BD (2 mentions)
Anti cd11c clone hl3, by BD (2 mentions)
Lsrfortessa flow cytometer, by BD (2 mentions)
Lsrii flow cytometer, by BD (2 mentions)
Anti cd3 clone 145 2c11, by BD (2 mentions)
Facscanto 2, by BD (2 mentions)
Automacs, by Miltenyi Biotec (2 mentions)
Anti cd3 cd28 antibody coated beads, by Thermo Fisher Scientific (2 mentions)
Miltenyi mouse pan t cell kits, by BD (2 mentions)
Anti cd25 clone 7d4, by BD (2 mentions)
Anti cd44 clone im7, by BD (2 mentions)
Cytoflex s, by Beckman Coulter (2 mentions)
Cellquest software, by BD (1 mentions)
16 protocols using anti cd8 clone 53 6
1
Quantification of Spinal Cord Immune Cells
2
In vivo Interaction Experiment with T. cruzi Infection
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For the in vivo interaction experiment, C57BL/6 mice (CD45.1) were irradiated at 900 Rads. Each irradiated animal received 10×106 bone marrow cells by i.v route, isolated from GREAT IFN-γ GFP reporter mice and REX3 CXCL10-BFP and CXCL9-RFP reporter mice (CD45.2). Twelve weeks after the transference, mice were infected with T. cruzi and, on day 12 after infection, spleens were harvested, then fixed with 1% of PFA, and labeled with DRAQ5 (BD, Pharmingen) and anti-CD8 (clone 53–67, BD). For experiments with REX3 mice, spleen cells were harvested on day 12 after infection, then fixed with 1% of PFA and labeled with: anti-CD8 (clone 53–67, BD), anti-CD11b (clone M1/70, ebioscience), anti-CD209a (clone MMD3, ebioscience), anti-CD317 (pDCA-1, Miltenyi Biotec), anti-CD11c (clone HL3, BD), and DRAQ5 (BD, Pharmingen). The samples were acquired from ImageStream Ammis, and we used the Ideas software to perform the analysis. The double cells were selected based on aspect ratio versus cell area [23 (link)].
3
Multicolor Flow Cytometry Analysis
4
Tumor Immune Cell Isolation Protocol
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Tumors were excised, weighed, and mechanically minced. Minced tumors were placed in gentle MACS Dissociator with Tumor Dissociation Kit for mouse tissues (Miltenyi Biotec, San Diego, CA, United States) to isolate immune and tumor cell subsets in accordance with the manufacturer’s directions. The cell suspension was passed through a 40-μm cell strainer (Falcon 352340) and washed twice. The responded cells were lysed with red blood cell lysis buffer (ACK) and incubated with mouse immunoglobulin G in FACS buffer for 15 min at 4°C. Tumor-infiltrating cells were stained with fluorochrome-conjugated anti–mouse antibodies, as well as appropriate isotype control antibodies. The following monoclonal antibodies and reagents were obtained from BD Bioscience (San Jose, CA, United States): anti-CD45 (30-F11, 1:200 dilution), anti-CD3 (clone 145-2c11, 1:200 dilution), anti-CD4 (GK1.5, 1:200 dilution), and anti-CD8 (clone 53–6.7, 1:200 dilution). Flow cytometry was carried out with LSRII flow cytometer (BD Biosciences, San Jose, CA, United States), and data were analyzed with FlowJo software (v.10.4; Tree Star, San Carlos, CA, United States).
5
Characterizing Lung Immune Cell Types
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Single-cell lung suspensions were prepared as previously described 34 (link). Innate immune cells were classified based on surface expression of the following markers: AMs: CD11chiCD11b−, PMNs: Ly6GhiCD11bhi, LMs: CD11c−Ly6G−F4/80+CD11b+, iMs: CD11c−Ly6G−F4/80+CD11b+Ly6Chi. The following mAbs were used for these investigations: anti-Ly6G (clone 1A8; BD), anti-Ly6C (clone AL-21; BD), anti-CD11b (clone M1/70; BD), anti-F4/80 (clone BM8; ebiosciences), and anti-CD11c (clone HL3; ATCC).
For intracellular cytokine staining (ICS), 2–4 × 106 lung cells were cultured for 6 h in medium containing Brefeldin A, either alone or supplemented with purified protein derivative (SSI). Cells were phenotyped according to their CD4 and CD8 surface expression (anti-CD4, clone RM4–5; BD), (anti-CD8, clone 53–6.7, BD) and staining intracellularly with anti-IFN-γ (clone XMG1.2, BD) and anti-TNF-α (clone XT-22, ATCC) Abs. Stained cells were acquired using a BD FACS Canto II instrument. Data analysis was carried out using BD FACSDiva and FACS Analyzer software.
For intracellular cytokine staining (ICS), 2–4 × 106 lung cells were cultured for 6 h in medium containing Brefeldin A, either alone or supplemented with purified protein derivative (SSI). Cells were phenotyped according to their CD4 and CD8 surface expression (anti-CD4, clone RM4–5; BD), (anti-CD8, clone 53–6.7, BD) and staining intracellularly with anti-IFN-γ (clone XMG1.2, BD) and anti-TNF-α (clone XT-22, ATCC) Abs. Stained cells were acquired using a BD FACS Canto II instrument. Data analysis was carried out using BD FACSDiva and FACS Analyzer software.
6
Flow Cytometric Analysis of Immune Cells
7
Isolation and Activation of Mouse CD4+CD25- T Cells
8
Quantifying Tumor-Infiltrating Immune Cells
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Tumor-bearing mice were sacrificed 3 days after the third treatment. The tumors were removed from the mice, embedded in an optimal cutting temperature compound (Sakura Finetek USA, Torrance, CA), and then cryosectioned to a thickness of 5 μm. The immune cells were detected with anti-CD4 (clone GK 1.5; BD Biosciences, San Diego, CA, USA) and anti-CD8 (clone 53-6.7; BD Biosciences) antibodies. The tumor-infiltrating immune cells were counted at a magnification of 150×. Three randomly chosen fields/samples from three mice were evaluated.
9
T Cell Depletion and Flow Cytometry
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At 3 days and 1 day before viral challenge, all cohorts of mice were treated intraperitoneally with 500 μg each of both anti-CD4 (clone GK1.5) and anti-CD8a (YTS 169.4) (BioXcell)52 (link)53 (link). Blood samples from each mouse were then assessed for the presence of CD4 and CD8 T cells by flow cytometry. Cells were fluorescently labelled with anti-CD4 (clone RM4-5, ebioscience) and anti-CD8 (clone 53-6.7, BD) at a dilution of 1:200 in FACS buffer (1% fetal bovine serum in PBS). Cells were then analysed using the BD FACSCalibur and flow plots created in FlowJo 8.7.
10
Multiparametric Flow Cytometry Analysis
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Blood or brain single-cell suspensions were washed in PBS supplemented with 1% FBS, 2 mM EDTA and 0.01% sodium azide, and incubated for 10 min at 4 • C with culture supernatant from the hybridoma cell line 2.4G2 to block Fc receptors. The cells were surface stained with fluorochrome-conjugated monoclonal antibody for 15 min at 4 • C. LIVE/DEAD fixable blue dye (Life Technologies) was used to exclude dead cells. The following antibodies were used: anti-CD45.1 (clone A20, BioLegend), anti-CD11b (clone M1/70, BD), anti-CD19 (clone 6D5, BioLegend), anti-CD3 (clone 145-2C11, BioLegend), anti-CD4 (clone RM4-5, BD), anti-CD8 (clone 53-6.7, BD or Biolegend), anti-FOXP3 (clone FJK-16s, eBioscience), anti-CD44 (clone IM7, BioLegend), anti-CD62L (clone MEL-14, BD), anti-CD127 (clone A7R34, BioLegend) and anti-KLRG1 (clone 2F1, eBioscience). OVA-specific CD8 + T cells were detected using H-2K b /SIINFEKL pentamers (ProImmune). Intracellular FoxP3 staining was performed using BioLegend FOXP3 Fix/Perm Buffer Set. Samples were acquired on LSRII Flow Cytometer (BD), and the data were analyzed using FlowJo software (TreeStar Inc.).
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