Cd8 percp

Manufactured by BD

Sourced in United States, Germany

The CD8-PerCP is a laboratory instrument used for the detection and analysis of CD8+ T cells in biological samples. It utilizes the principle of flow cytometry to quantify the expression of the CD8 surface marker on cells, which is a key indicator of T cell function and differentiation.

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Lab products found in correlation

Cd3 fitc, by BD (12 mentions) Facscanto 2, by BD (10 mentions) Cd4 percp, by BD (8 mentions) Facsdiva software, by BD (7 mentions) Cd4 fitc, by BD (7 mentions) Cd3 apc, by BD (5 mentions) Facscanto 2 flow cytometer, by BD (5 mentions) Golgiplug, by BD (4 mentions) Facscanto, by BD (4 mentions) Cd4 pe, by BD (4 mentions) Cytofix cytoperm, by BD (4 mentions) Facscalibur, by BD (4 mentions) Cd25 apc, by BD (3 mentions) Ccr7 pe cy7, by BD (3 mentions) Lsrii flow cytometer, by BD (3 mentions) Facs lysing solution, by BD (3 mentions) Cd28 pe, by BD (3 mentions) Cd95 apc, by BD (3 mentions) Cd19 apc h7, by BD (3 mentions) Ifn γ pe, by BD (3 mentions)

53 protocols using cd8 percp

Immune Profiling of COVID-19 Patients

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Left-over blood samples for clinical examination from 32 COVID-19 patients and 37 healthy volunteers were collected for analysis. All the samples were processed in the Clinical Lab of Wuhan Union Hospital based on uniform standard procedure. Briefly, peripheral blood mononuclear cells (PBMCs) and plasma were isolated by density gradient centrifugation. Fresh separated PBMCs were stained for flow cytometry, and plasma samples were used for cytokine detection.
Flow cytometry was performed as described previously (9 (link)). Briefly, 1 × 10⁶ PBMCs were stained with indicated antibodies in the dark at room temperature for 20 min. After several washes, the cells were analyzed within 1 h. All samples were detected by BD FACS Canto II Flow Cytometry System and analyzed with the BD FACS Diva Software. Antibodies used for flow cytometry included FITC-CD3 (clone: HIT3a), PE-PD-1 (clone: EH12.1), PE/Cy7-CD56 (clone: B159), APC-CD244 (clone: 2-69), APC/Cy7-CD45 (clone: 2D1), BV421-CD16 (clone: 3G8), BV421-CD4 (clone: RPA-T4), PerCP-CD8 (clone: RPA-T8), PE/Cy7-CD27 (clone: M-T271), and all of these were purchased from BD Pharmingen.

Li M., Guo W., Dong Y., Wang X., Dai D., Liu X., Wu Y., Li M., Zhang W., Zhou H., Zhang Z., Lin L., Kang Z., Yu T., Tian C., Qin R., Gui Y., Jiang F., Fan H., Heissmeyer V., Sarapultsev A., Wang L., Luo S, & Hu D. (2020). Elevated Exhaustion Levels of NK and CD8+ T Cells as Indicators for Progression and Prognosis of COVID-19 Disease. Frontiers in Immunology, 11, 580237.

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Detecting lymphocyte activation in cardiac transplants

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For the detection of cell activation in vitro, purified CD19⁺ B cells were cultured for 2 days and incubated with APC-CD25 and PE-CD69 (BD Bioscience) for 30 min at 4 °C in dark.
For the detection of lymphocytes in the model, peripheral blood, fresh cardiac grafts and recipient spleens were obtained on 4 days after cardiac transplantation, before the allo-graft failure. Fresh recipient cardiac grafts were digested in phosphate-buffered saline supplemented with 1% heat-inactivated fetal bovine serum with collagenase 1 and DNase for 60 min at 37 °C, before pressing through a 200 mesh nylon screen. The collected cells were isolated by gradient density centrifugation using ficoll-paque premium (Cytiva, Washington, USA). Collected lymphocytes were stained with fluorescently labeled antibodies. Pacificblue-CD45, PE-CD45R, fluorescein isothiocyanate (FITC)-CD3, APCcy7-CD4, Percp-CD8, PEcy7-CD11b, APC-CD161, FITC-immunoglobulin (Ig)M, and Percpcy5.5-IgG antibodies used for flow cytometry were from BD Biosciences.
Detection was determined by a flow cytometer (CytoFLEX; Beckman Coulter, Fullerton, CA, USA) and data were analyzed by FlowJo (Tree Star, Ashland, OR, USA).

Zhang Y., He J., Yang Z., Zheng H., Deng H., Luo Z., Sun Q, & Sun Q. (2023). Preventative effect of TSPO ligands on mixed antibody-mediated rejection through a Mitochondria-mediated metabolic disorder. Journal of Translational Medicine, 21, 295.

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Intracellular Cytokine Profiling in Lymphocytes

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To detect intracellular cytokine production, 5 × 10⁶ splenocytes or pulmonary lymphocytes were incubated at 37°C with 10 μM mixed peptides plus 1 μg/ml anti-CD28 mAb for 10 h, and treated with 10 μg/ml Brefeldin A (BD Pharmingen) for an additional 5 h. 1 × 10⁶ cells were first stained with surface markers of PE-Cy7-CD4 or PerCP-CD8(BD Pharmingen) for 30 min, then fixed and permeabilized by Cyto Fix/Perm (BD Pharmingen) for 30 min. Intracellular staining was performed using FITC-IFN-γ, PE-IL-2, APC-TNF-α Abs (BD Biosciences). Flow cytometric data were acquired using a FACSCanto II (BD Biosciences) and analyzed using FlowJo software (TreeStar, San Carlos, CA).

Wu M., Zhao H., Li M., Yue Y., Xiong S, & Xu W. (2017). Intranasal Vaccination with Mannosylated Chitosan Formulated DNA Vaccine Enables Robust IgA and Cellular Response Induction in the Lungs of Mice and Improves Protection against Pulmonary Mycobacterial Challenge. Frontiers in Cellular and Infection Microbiology, 7, 445.

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Phenotyping of Immune Cells by Flow Cytometry

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Antibodies for surface markers staining of DCs and T cells were obtained from BD Biosciences, New York, USA (anti-human CD3-PE, CD3-FITC, CD8-PerCP, CD8-APC, CD56-PE, NKG2D-APC, CD4-FITC, CD4-PerCP, CD107a-FITC, CD25-APC, CD45RO-FITC, CD27-PerCPCY5.5, CD57-APC, CCR7-PE, CD14-APC, CD80-PE, CD83-APC, CD86-FITC, HLA-DR-FITC). Antibodies for intracellular proteins staining were also obtained from BD Biosciences (anti-human IFNγ-APC, TNFα-PECY7, granzyme B-FITC, FoxP3-PE). The intracellular staining was performed by fixing and permeabilizing cells with Cytofix/Cytoperm (BD Biosciences). The experiments were performed by using FACS CantoII (BD Biosciences) flow cytometer, and data were analyzed by using the Flowjo software.

Han Y., Wu Y., Yang C., Huang J., Guo Y., Liu L., Chen P., Wu D., Liu J., Li J., Zhou X, & Hou J. (2017). Dynamic and specific immune responses against multiple tumor antigens were elicited in patients with hepatocellular carcinoma after cell-based immunotherapy. Journal of Translational Medicine, 15, 64.

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Comprehensive Immune Cell Profiling by Flow Cytometry

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Peripheral blood mononuclear cells (PBMC) were isolated by density centrifugation with Lymphoprep (Axis-Shield). PBMC or whole blood samples were stained with the following fluorochrome-conjugated monoclonal antibodies: CD3-efluor605, CD4-efluor450, CD27-APC-efluor780, HLA-DR-efluor780, CD45RA-efluor605, FOXP3-PE (eBioscience), CD4-APC-H7, CD8-Percp, CD8-PE-Cy7, CD31-AF647, CD45RO-FITC, CD45RO-PE-Cy7, CCR7-PE-Cy7, Ki-67-Percp-cy5.5, CTLA-4-BV421 (BD Biosciences), PD-1-PE, CD28-AF700 (Biolegend), and CD161-PE (Miltenyi Biotec). Intracellular staining for FOXP3, Helios, Ki-67, and CTLA-4 was performed after cells were permeabilized with a FOXP3 staining buffer set according to instructions of the manufacturer (eBioscience). Whole blood samples were treated with BD lysing solution according to the instructions of the manufacturer (BD Biosciences). Stained samples were analyzed on a LSR-II flow cytometer (BD Biosciences). Analysis was performed with Kaluza Flow Analysis Software (Beckman Coulter). Absolute numbers of CD3+ T cells, CD4+ T cells, CD8+ T cells, B cells, and NK cells were determined according to the MultiTest TruCount method (BD Biosciences), as described by the manufacturer. TruCount samples were measured on a FACSCanto-II (BD Biosciences) and analyzed with FACSCanto Clinical Software (BD Biosciences).

van den Brom R.R., van der Geest K.S., Brouwer E., Hospers G.A, & Boots A.M. (2018). Enhanced expression of PD-1 and other activation markers by CD4+ T cells of young but not old patients with metastatic melanoma. Cancer Immunology, Immunotherapy, 67(6), 925-933.

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T-cell Stimulation and Cytotoxicity Assay

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On day 0, 7, and 14, the PBMCs from the T‐cell stimulation assay were analyzed in flow‐cytometry by anti‐CD3‐FITC, CD8‐PerCP, CD45RO‐PE, CD45RA‐APC‐H7, and CD69‐APC antibodies (BD, Heidelberg, Germany) with a FACS Canto II.
Cytotoxicity of effector cells that were stimulated for 14 days was tested by measuring LDH release with the CytoTox 96 Non‐Radioactive Cytotoxicity Assay (Promega, Madison, Wisconsin) following incubation with peptide loaded T2/mHLA‐A*24:02 cells at a ratio of 5:1. The cells were incubated for 4 hours at 37°C and LDH release was measured according to kit instructions.

Pump W.C., Schulz R., Huyton T., Kunze‐Schumacher H., Martens J., Hò G.T., Blasczyk R, & Bade‐Doeding C. (2019). Releasing the concept of HLA‐allele specific peptide anchors in viral infections: A non‐canonical naturally presented human cytomegalovirus‐derived HLA‐A*24:02 restricted peptide drives exquisite immunogenicity. Hla, 94(1), 25-38.

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Murine Splenocyte Isolation and Flow Cytometry

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Splenocytes from Balb/c mice were isolated and erythrocytes were lysed by treatment with FACS lysing solution (BD Biosciences, USA), prepared according to manufacturer’s instructions. Cells were washed in PBS and suspended in 1% (w/v) bovine serum albumin (Sigma) prepared in PBS. Approximately 10⁶ splenocytes were pelleted and stained for 30 min at 4°C in the dark with 20 μL of fluorescent monoclonal antibodies against: CD3-PE, CD4-FITC or CD4-Alexa Fluor 647, CD8-PerCP, B220-APC and CD45RB-FITC (BD Biosciences), previously titrated and mixed. Splenocytes were washed twice, suspended in 300 μL of PBS and read in a BD Accuri C6 Flow Cytometer (BD, USA). Cell populations were analyzed offline using FlowJo software (Tree Star, USA). For the in vivo cytotoxicity analysis, CFSE-stained cells were readily analyzed without additional markers.

Gonçalves A.J., Oliveira E.R., Costa S.M., Paes M.V., Silva J.F., Azevedo A.S., Mantuano-Barradas M., Nogueira A.C., Almeida C.J, & Alves A.M. (2015). Cooperation between CD4+ T Cells and Humoral Immunity Is Critical for Protection against Dengue Using a DNA Vaccine Based on the NS1 Antigen. PLoS Neglected Tropical Diseases, 9(12), e0004277.

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Immunosenescent Cell Analysis in Whole Blood

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UCLA Immune Assessment Core performed the analysis of immunosenescent cells for 144 whole blood samples, as described previously [71 (link)]. Briefly, Total CD3 + T-cells, CD4 + T-cells, CD8 + T-cells, CD19 + B-cells, and CD56 + /CD16 + NK-cells were enumerated in EDTA whole blood with the BD Multitest 6-color TBNK reagent and BD Trucount tubes following the manufacturer’s instructions, acquired on a BD FACSCanto II and analyzed with the BD FACSCanto Software. CD8 + T-cell sub setting was performed by staining 50 μl of EDTA whole blood with CD3 FITC, CD8 PerCP, CD28 PE, and CD95 APC (BD) for 10 min, followed by BD FACS Lysing used according to the manufacturer’s instructions. At least 10,000 lymphocyte events per sample were acquired and analyzed using DIVA 8.0 software on BD FACSCanto II.

Luo Q., Dwaraka V.B., Chen Q., Tong H., Zhu T., Seale K., Raffaele J.M., Zheng S.C., Mendez T.L., Chen Y., Carreras N., Begum S., Mendez K., Voisin S., Eynon N., Lasky-Su J.A., Smith R, & Teschendorff A.E. (2023). A meta-analysis of immune-cell fractions at high resolution reveals novel associations with common phenotypes and health outcomes. Genome Medicine, 15, 59.

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Single B Cell Sorting for Antigen-Specific Isolation

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Single B cell sorting was followed as previously described (3 (link), 71 (link)). Briefly, PBMCs from donor CBJC438 were incubated with an antibody cocktail consisting of CD19-PE-Cy7, CD8-PerCP, IgM-PE-Cy5, IgG-FITC (all from BD Biosciences), CD20-ECD (Beckman), CD3–Pacific Orange (Invitrogen), CD14–eFluor 450 (eBioscience), BG505-APC (purified in laboratory), and LIVE/DEAD Fixable Dead Cell Stain Kit (Invitrogen) to exclude dead cells. The PBMCs were first stained with 50 μl of phosphate-buffered saline (PBS) with LIVE/DEAD at 4°C in the dark for 30 min and then stained with 50 μl of PBS with an antibody cocktail for 1 hour at 4°C in the dark. The stained cells were washed and resuspended in PBS and then passed through a 70-μm cell mesh (BD Biosciences). Antigen-specific single B cells were gated as CD19⁺CD20⁺CD8⁻CD3⁻CD14⁻IgM⁻IgG⁺BG505⁺ and sorted into a 96-well PCR plate containing 20 μl of lysis buffer: 5 μl of 5× first-strand buffer, 0.5 μl of RNaseOUT, 1.25 μl of 0.1 M dithiothreitol (all from Invitrogen), and 0.0625 μl of IGEPAL (Sigma-Aldrich) per well. The sorted plate was snap-frozen on dry ice and stored at −80°C before reverse transcription (RT) reaction.

Kumar S., Ju B., Shapero B., Lin X., Ren L., Zhang L., Li D., Zhou Z., Feng Y., Sou C., Mann C.J., Hao Y., Sarkar A., Hou J., Nunnally C., Hong K., Wang S., Ge X., Su B., Landais E., Sok D., Zwick M.B., He L., Zhu J., Wilson I.A, & Shao Y. (2020). A VH1-69 antibody lineage from an infected Chinese donor potently neutralizes HIV-1 by targeting the V3 glycan supersite. Science Advances, 6(38), eabb1328.

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Detecting IL-21 Production in PBMC Subsets

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Patient's PBMC were stimulated for 3 days at 37°C, 5% CO₂ and 95% humidity with CSFE-labeled, irradiated (40 Gy) donor PBMCs, depleted for CD3⁺ T cells, in culture medium. At the end of day 2 monensin and brefeldin A (GolgiStop and GolgiPlug, BD Biosciences) were added for 16 h in a concentration of 1:1,500 and 1:1,000, respectively, to allow the measurement of intracellularly accumulated cytokines in PBMCs. Thereafter, intracellular IL-21 was measured, and surface marker staining was used to investigate which subsets produced these cytokines. Monoclonal antibodies used for surface marker staining and intracellular cytokine staining were CD3 BV510 (BioLegend), CD4 BV421 (BioLegend), CD8 PerCP (BD Biosciences), CXCR5 APC (BD Biosciences), PD-1 APC-Cy7 (BioLegend), and IL-21 PE (Biolegend).

van Besouw N.M., Yan L., de Kuiper R., Klepper M., Reijerkerk D., Dieterich M., Roelen D.L., Claas F.H., Clahsen-van Groningen M.C., Hesselink D.A, & Baan C.C. (2019). The Number of Donor-Specific IL-21 Producing Cells Before and After Transplantation Predicts Kidney Graft Rejection. Frontiers in Immunology, 10, 748.

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