The following antibodies were purchased from BD Pharmingen, unless otherwise noted: BV421-conjugated anti-CD3 (#562426); BV421-conjugated anti-CD4 (BioLegend, #300532); BV421-conjugated anti-CD56 (BD Horizon, #562751); BV421-conjugated anti-CD86 (#562432); BV421-conjugated anti-HLA-DR (BD Horizon, #562804); BV421-conjugated IgG1, κ Isotype Control (BioLegend, #400158); BV421-conjugated IgG2a, κ Isotype Control (BD Horizon, #562439); BV421-conjugated IgG2b, κ Isotype Control (BioLegend, #400342); FITC-conjugated anti-CD4 (#555346); FITC-conjugated anti-CD14 (#555397); FITC-conjugated anti-CD15 (#562370); FITC-conjugated anti-CD19 (#555412); FITC-conjugated anti-CD25 (#555431); FITC-conjugated anti-CD69 (#555530); FITC-conjugated IgG1, κ Isotype Control (#555748); FITC-conjugated IgG2a, κ Isotype Control (#555573); PE-conjugated anti-CD8 (#555367); PE-conjugated anti-CD11b (BioLegend, #301406); PE-conjugated anti-Cd11c (#555392); PE-conjugated IgG1, κ Isotype Control (#555749); APC-conjugated anti-CD3 (#555335); APC-conjugated anti-CD11a (R&D Systems, #FAB3595A); APC-conjugated anti-HLA-DR (#559866); APC-conjugated IgG1, κ Isotype Control (#555751); and APC-conjugated IgG2a, κ Isotype Control (BioLegend, #400220).
Pe conjugated anti cd8
Manufactured by BioLegend
Sourced in United States
PE-conjugated anti-CD8 is a fluorochrome-labeled monoclonal antibody that binds to the CD8 receptor on the surface of T cells. It is designed for use in flow cytometry applications to identify and quantify CD8+ T cells in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Fitc conjugated anti cd4, by BioLegend (3 mentions)
Fitc conjugated anti cd19, by BioLegend (2 mentions)
Apc cy7 conjugated anti cd4, by BioLegend (2 mentions)
Bv421 conjugated anti cd4, by BioLegend (1 mentions)
Fitc conjugated anti cd14, by BioLegend (1 mentions)
Fitc conjugated anti cd25, by BioLegend (1 mentions)
Fitc conjugated anti cd69, by BioLegend (1 mentions)
Pe conjugated anti cd11b, by BioLegend (1 mentions)
Apc conjugated anti hla dr, by BioLegend (1 mentions)
Bv421 conjugated anti cd3, by BioLegend (1 mentions)
Facs lysing solution, by BD (1 mentions)
Minicollect tube, by Greiner (1 mentions)
Fluorescein isothiocyanate (fitc), by BioLegend (1 mentions)
Pe conjugated anti il 17, by BioLegend (1 mentions)
Pe conjugated anti nk1, by BioLegend (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Apc conjugated anti cd4, by BioLegend (1 mentions)
Lsrfortessa, by BD (1 mentions)
Cyan adp analyzer, by Beckman Coulter (1 mentions)
Apc conjugated anti c kit, by BioLegend (1 mentions)
10 protocols using pe conjugated anti cd8
1
Multiparameter Flow Cytometry Panel
2
Isolation and Characterization of Lymphocyte Subsets
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MSLs were isolated as published previously.23 (link) For the separation of MPBLs, peripheral blood was collected with MiniCollect Tubes (Greiner Bio-One, Austria), and erythrocytes were lysed with lysing solution (BD FACSTM Lysing Solution). Following antibody staining, the subpopulation of lymphocytes was measured by flow cytometry. Mice monoclonal antibodies (mAbs) were used for multi-parameter flow cytometric analysis: allophycocyanin (APC)-conjugated anti-CD3, phycoerythrin (PE)-conjugated anti-NK1.1, fluorescein isothiocyanate (FITC)-conjugated anti-CD19, FITC-conjugated anti-CD4, PE-conjugated anti-CD8, and PE-conjugated anti-IL-17 were obtained from BioLegend (London United Kingdom); and PE-conjugated anti-CD25 and APC-conjugated anti-FoxP3 were provided by ImmunoTools (Friesoythe; Germany). Lymphocytes were incubated with antibodies in 1% (w/v) bovine serum albumin/phosphate-buffered saline (PBS) at 4 °C for 1 h in the dark. Cells were washed twice with PBS and analyzed by flow cytometry (BD Biosciences). WinMDI2.9 software was used to analyze the percentages of different lymphocyte subpopulations.
3
Multiparametric Phenotyping of Thymic Cells
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Single-cell suspensions of thymocytes and TECs cells were stained with one or more of the following fluorochrome-conjugated antibodies: Allophycocyanin (APC)-conjugated anti-CD4, phycoerythrin (PE)-conjugated anti-CD8, PerCP/Cy5.5-conjugated anti-epithelial cellular adhesion molecule (EpCAM) 1, fluorescein isothiocyanate-conjugated anti-major histocompatibility complex (MHC) II, PE-conjugated anti-ulex europeus lectin (UEA) and APC-conjugated anti-CD45.1 (BioLegend, BD Biosciences, USA). The samples were analyzed on a FACSCalibur flow cytometer (Becton and Dickinson, USA). Data analysis was conducted using FlowJo software package (TreeStar, Inc., Ashland, USA).
4
Multiparameter Flow Cytometry of Hematopoietic Cells
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The whole blood was stained with APC-conjugated anti-Ter119 and PE-conjugated anti-CD61 for erythrocyte and platelet analysis. White blood cells after RBC lyses were stained with PE-conjugated anti-CD8, PE-Cy7–conjugated anti-B220, APC-conjugated anti–Mac-1, APC-Cy7–conjugated anti-CD4, and Pacific blue (PB)–conjugated anti-Gr1 (BioLegend). Single cell suspensions from BM and spleen were stained with lineage-marker cocktail followed by FITC-conjugated anti-CD41, PE-conjugated anti-CD150, PE-Cy7–conjugated anti-CD48, APC-conjugated anti–c-Kit, BV421-conjugated anti–Sca-1, and streptavidin-APC-Cy7. For progenitor analysis, cells were stained with lineage-marker cocktail followed by FITC-conjugated anti-CD34 (BD), PE-conjugated anti-CD16/32, APC-conjugated anti–c-Kit, BV421-conjugated anti–Sca-1, and streptavidin-APC-Cy7 antibodies (BioLegend). For megakaryocytes and erythrocyte precursor analysis, cells were stained with FITC-conjugated anti-CD42c (Emfret), Dylight-647–conjugated anti-αIIbβ3 (CD41/61; Emfret), PE-conjugated anti-CD71, and APC-conjugated anti-Ter119. Analyses were performed on an LSRFortessa (BD) or CyAn ADP Analyzer (Beckman Coulter), and results were analyzed with FlowJo software.
5
Intracellular IFN-γ and Granzyme B Staining
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For intracellular IFN-γ and Granzyme B staining, CD8+ T cells (2 × 105 cells per well) were seeded in 24-well plates and treated with or without anti-CD3, anti-CD28 (Biolegend), or monoclonal anti-mouse B7-H5 antibody. After 72 h, CD8+ T cells were incubated with Brefeldin A (Biolegend) for 10 h. Subsequently, cells were surface stained with PE-conjugated anti-CD8 (Biolegend), followed by intracellular staining for APC-conjugated anti-IFN-γ (Invitrogen, Carlsbad, CA, USA) and PE/Cy7-conjugated anti-Granzyme B (Biolegend).
6
Quantifying Tumor-Infiltrating Lymphocytes and Cell Apoptosis
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On the seventh day after the injection of SCC7 cells, the mice were sacrificed, and the tumour tissues were extracted to make a single‐cell suspension. The mouse TILs were isolated from the tumours using the tumour lymphocyte infiltration kit (Solarbio, Beijing, China), stained with APC‐conjugated anti‐CD3, PE‐conjugated anti‐CD8 and PerCP‐conjugated anti‐CD4 (Biolegend, San Diego, CA, USA), and detected using flow cytometry.
For the apoptosis assay, SCC7 cells were harvested and detected using Annexin V‐PE/7‐AAD staining assay and flow cytometry.
For the apoptosis assay, SCC7 cells were harvested and detected using Annexin V‐PE/7‐AAD staining assay and flow cytometry.
7
Isolation and Analysis of Tumor-Infiltrating Lymphocytes
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Fresh tumors obtained from mice were cut into pieces (3–5 mm3) and treated with collagenase I (1 µg/mL) and collagenase IV (1 µg/mL) for 1.5 hours at 37°C. The tissue homogenates were filtered using a 70 µm cell strainer (Falcon) before centrifugation. The samples were subjected to density gradient centrifugation using Ficoll-Paque PLUS density gradient media. The supernatant was discarded and the cells were suspended in an appropriate medium. The samples were centrifuged to obtain tumor-infiltrating lymphocytes (TILs). The TILs were stained with FITC-conjugated anti-CD3 (BioLegend), APC-Cy7-conjugated anti-CD4 (BioLegend), PE-conjugated anti-CD8 (BioLegend), PerCP-conjugated anti-CD45.1 (BioLegend), and APC-conjugated anti-interferon gamma (IFNγ) (BioLegend) antibodies. The stained samples were analyzed using a BD FACS Canto II (BD Biosciences) cytometer.
8
Isolation and Culture of T-cell Subsets
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Whole blood samples were collected from each subject by venipuncture, and density gradient centrifugation was used to extract PBMCs. CD4+ T cells (CD3+CD4+), CD8+ T cells (CD3+CD8+), monocytes (CD3−CD14+), natural killer (NK) cells (CD3−CD56+), and B cells (CD3−CD19+) of HCs were sorted from PBMCs using a BD FACS Aria flow cytometer. The following monoclonal antibodies (mAbs, Biolegend) were used: PerCP-conjugated anti-CD3, FITC-conjugated anti-CD4, PE-conjugated anti-CD8, PE-CY7-conjugated anti-CD14, PE-CY7-conjugated anti-CD56 and PerCP-conjugated anti-CD19. STEMCELL was used to sort CD8+ T cells from HIV-infected patients, and the sorting purity was detected by flow cytometry. CD8+ T cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 supplemented with 10% fetal bovine serum (FBS) (HyClone), 1% penicillin–streptomycin (Gibco) and IL-2 [30 (link), 31 (link)] (30 U/ml, Sigma). Transfection of siRNA and controls (Invitrogen) was performed with Lipofectamine™ RNAiMAX Transfection Reagent (Invitrogen). Briefly, cells were transfected with 100 pmol of eIF3d siRNA or 75 pmol of eIF3d siRNA plus 75 pmol of SOCS-7 siRNA. Transfection efficiency was measured by real-time PCR after 48 h of transfection.
9
Characterizing Cell Surface Markers
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Cells were washed with ice-cold PBS and incubated for 15 min with the Fc-receptor-blocking antibodies anti-CD16/anti-CD32 (BD Biosciences Pharmingen). After washing with PBS + 2% FCS, cells were stained for 20 min. HPB-ALL cells were stained with the phycoerythrin (PE)-conjugated anti-CD127/IL7Rα (Biolegend, cat. no. 351342) and human peripheral blood mononuclear cells (PBMCs) were stained with phycoerythrin (PE)-conjugated anti-CD8 (Biolegend, cat. No. 344706), allophycocyanin (APC)-conjugated anti-CD127/IL7Rα (Biolegend, cat. no. 351315) and a Zombie Aqua™ viability dye (BioLegend). Cells were washed twice, fixed with 0.37% formaldehyde and analyzed on a BD LSR Fortessa X20 with DIVA software (BD Biosciences, v9.0). Results were further analyzed with the FlowJo software package (BD Biosciences, v10.0). An example of the gating strategies is illustrated in Supplementary Figure 4
10
Isolation of CD8+ T Cells from Murine Spleen
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CD8+ T cells were isolated from the spleen of BALB/c mice using anti-PE MicroBeads (Miltenyi Biotec) and PE-conjugated anti-CD8 (Biolegend) according to the manufacturer’s protocol.
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