Percp cy5.5 anti cd4 clone okt4

Manufactured by BioLegend

Sourced in United States

PerCP-Cy5.5 anti-CD4 (clone OKT4) is a fluorescently-labeled monoclonal antibody that binds to the CD4 antigen expressed on the surface of T helper cells. It is a useful tool for the identification and enumeration of CD4+ T cells in flow cytometry applications.

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CD4-PerCp-Cy5.5 clone OKT4 CD4-PerCP (clone OKT4, PerCP/Cy5.5 anti-CD4 Anti-CD4 (clone OKT4, PerCP-Cy5.5-CD4 Anti-CD4-PerCP/Cy5.5 CD4 (clone OKT4; CD4-PerCP/Cy5.5 Anti-CD4 (OKT4, CD4 (OKT4,

7 protocols using percp cy5.5 anti cd4 clone okt4

Multiparameter Flow Cytometric Analysis of T Cells

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Multiparameter flow cytometry was performed using peripheral blood, as described previously (Ma et al., 2012 (link); Xia et al., 2009 (link); Zhang et al., 2017b (link); Zheng et al., 2014a (link)). For the frequency of HLA-DR⁺CD8⁺ T cells, blood was treated with lysing buffer (BD Biosciences, CA, USA) for 10 min and then incubated with a mixture of flow cytometry antibodies at 4 °C for 30 min. For staining with ki67, surface-labeled cells were further treated with fixation and permeabilization solution (BD Biosciences, CA, USA), followed by perm/wash buffer (BD Biosciences, CA, USA). Cross-reactive flow cytometry human antibodies anti-CD3 APC-Cy7 (clone SP34-2), anti-CD8 PE-Cy7 (clone RPA-T8), anti-HLA-DR APC (clone G46-6), and anti-ki67 PE (B56) were purchased from BD Pharmingen (Franklin Lakes, NJ, USA). Anti-CD4 PerCP-Cy5.5 (clone OKT4) was obtained from Biolegend (San Diego, CA, USA). Flow cytometry acquisition was performed on a BD FACSVerse^TM flow cytometer (BD, Franklin Lakes, NJ, USA) and flow cytometric data analysis was performed using FlowJo vX.0.7 (TreeStar, Ashland, OR, USA).

Zhang M.X., Song T.Z., Zheng H.Y., Wang X.H., Lu Y., Zhang H.D., Li T., Pang W, & Zheng Y.T. (2019). Superior intestinal integrity and limited microbial translocation are associated with lower immune activation in SIVmac239-infected northern pig-tailed macaques (Macaca leonina). Zoological Research, 40(6), 522-531.

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Expanding Cryo-preserved PBMCs with Cytokines

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Cryo-preserved PBMCs were thawed, and washed with PBS. Cells were labeled with 5 μM CellTrace Violet (Thermo Fisher Scientific) in PBS at 37°C in a dark water bath for 20 minutes and quenched for 5 min over ice in the presence of 10% FBS. Cell Trace-labeled PBMCs were washed and resuspended (2.5 × 10⁶ cells per ml) in R10 with 30 ng/ml recombinant human IL-15, and 30 ng/ml recombinant human IL-7. Stimulated cells were fed with fresh medium supplemented with 60 ng/ml recombinant human IL-15, and 60 ng/ml recombinant human IL-7 (final concentration of 30 ng/ml for each cytokine) at day 4 and were harvested at day 7. After stimulation, cells were washed and stained with the following combination of monoclonal antibodies: anti-CD3-BUV395 (clone SP34-2), anti-CD8-BUV563 (clone RPA-T8), anti-Ki-67-AL700 (clone B56), all from BD Biosciences; anti-CD4-PerCP-Cy5.5 (clone OKT4) from Biolegend; LIVE/ DEAD Fixable Near-IR Dead Cell Stain from Thermo Fisher Scientific. Samples were collected on a FACSymphony system (BD Biosciences) and analyzed in FlowJo (version 9.9.6, TreeStar).

Pino M., Pereira Ribeiro S., Pagliuzza A., Ghneim K., Khan A., Ryan E., Harper J.L., King C.T., Welbourn S., Micci L., Aldrete S., Delman K.A., Stuart T., Lowe M., Brenchley J.M., Derdeyn C.A., Easley K., Sekaly R.P., Chomont N., Paiardini M, & Marconi V.C. (2021). Increased homeostatic cytokines and stability of HIV-infected memory CD4 T-cells identify individuals with suboptimal CD4 T-cell recovery on-ART. PLoS Pathogens, 17(8), e1009825.

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Multiparameter Flow Cytometry of Immune Cells

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Six-parameter ow cytometric analysis was performed on whole blood, BM-, LN-and spleen-derived cells according to standard procedures using a panel of mAbs purchased from BD Biosciences (Pharmingen, San Diego, CA) as follows: anti-CD3-APC-Cy7 (clone SP34), anti-CD8-PE (clone RPA-T8), anti-Ki-67-PE-Cy7 (clone B56), anti-CD14-PE (clone M5E2), anti-CD16-FITC (clone 3G8), and anti-HLA-DR-APC (clone G46-6). The anti-CD4-PerCP-Cy5.5 (clone OKT4) was purchased from Biolegend (San Diego, CA, USA). Isotype antibody was used for a negative control of CD4, Ki67, HLA-DR, CD14 and CD16 expression. Intracellular staining for Ki-67 was performed at room temperature for 30 minutes following permeabilization with cyto x/cytoperm (BD Bioscience). Flow cytometric acquisition and analysis were performed on a BD Verse cytometer driven by the FACS Verse software (BD Biosciences). Analysis of the acquired data was performed using FlowJo software (TreeStar, Ashland, OR, USA) and graphs were prepared using Prism version 6.0 (GraphPad).

Liu H., Liu J., Meng F., Xu X., Wang Y., Xian Q., Zhou R., Xiao Q., Huang Z., Zhou L., Li J., Li X., Wang X., Ho W., & Zhuang K. (2020). CD4+ T Cell Depletion Does Not Affect the Level of Viremia in Chronically SHIVSF162P3N-Infected Chinese Cynomolgus Monkeys.

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Efficient EV Uptake Quantification

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After incubation with EVs, the PBMCs were washed three times in FACS buffer (0.5% BSA in PBS) and then stained with fluorescent labeled monoclonal antibodies from Biolegend: APC anti-human CD19 (clone HIB19), FITC anti-mouse/human CD11b (clone M1/70), FITC anti-human CD14 (clone M5E2), PerCP/Cy5.5 anti-human CD25 Antibody (clone BC96), PE/Cy7 anti-human HLA-DR Antibody (clone L243), and Pacific Blue™ anti-human CD3 (clone HIT3a) or PerCP/Cy5.5 anti-CD4 (clone OKT4), and PE/cy7 anti-human CD8 (clone RPA-T8), according to manufacturer’s recommendations. In Fig. 4, we used additional antibodies including BV422 anti-human CD80 (clone 2D10), Alexa Fluor 700 CD15 (cloneW6D3), FITC anti-human CD14 (M5E2), and AmCyan CD11b (clone M1/70). After antibody incubation, cells were washed two times in FACS buffer. Flow cytometry data was collected on a FACS Canto II (BD, Franklin Lakes, NJ). Dead cells were excluded from analysis using forward and sideward scattering gating. Live cells were gated based on cell surface markers CD11b, CD14, CD19, CD4 and CD8 or CD3 to identify monocytes, B cells, and T cells, respectively. For Fig. 4, percent monocytes are shown as CD14⁺CD15⁻ cells gated on CD11b⁺ population. The cell populations were then also gated for PKH to identify cells that internalized the labeled EVs. The flow cytometry data was analyzed with FlowJo software (Tree Star, Inc.).

Eitan E., Green J., Bodogai M., Mode N.A., Bæk R., Jørgensen M.M., Freeman D.W., Witwer K.W., Zonderman A.B., Biragyn A., Mattson M.P., Noren Hooten N, & Evans M.K. (2017). Age-Related Changes in Plasma Extracellular Vesicle Characteristics and Internalization by Leukocytes. Scientific Reports, 7, 1342.

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Sorting and Expansion of Th1, Th1/Th17, and Th17 Cells from RRMS Patients

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Fresh peripheral blood samples from de-identified RRMS patients were collected under the approvement by the Northwestern University Institutional Review Board. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using ISOLYMPH (Cat #759050, CTL). The PBMCs were stained with Aqua LIVE/DEAD fixable staining reagents (Cat #L34957, Life Technologies), human FcR blocking reagent (Cat # 130-059-901, Miltenyi Biotec), APC anti-CD3 (clone OKT3, BioLegend), Percp-cy5.5 anti-CD4 (clone OKT4, BioLegend) PE-Cy7 anti-CD45RA (clone HI100, BioLegend), BV650 anti-CD45RO (clone UCHL1, BD Biosciences), Pacific Blue anti-CXCR3 (clone G025H7, BioLegend) and BB515 anti-CCR6 (clone 11A9, BD Biosciences). Then CD3⁺CD4⁺CD45RA⁻CD45RO⁺CCR6⁻CXCR3⁺ (Th1), CD3⁺CD4⁺CD45RA⁻CD45RO⁺CCR6⁺CXCR3⁺ (Th1/Th17) and CD3⁺CD4⁺ CD45RA⁻CD45RO⁺CCR6⁺CXCR3⁻ (Th17) cells were sorted. After sorting, the cells were amplified by stimulated with anti-CD3/CD28 Dynabeads for 7 days.

Qian Y., Arellano G., Ifergan I., Lin J., Snowden C., Kim T., Thomas J.J., Law C., Guan T., Balabanov R.D., Kaech S.M., Miller S.D, & Choi J. (2021). ZEB1 promotes pathogenic Th1 and Th17 cell differentiation in multiple sclerosis. Cell reports, 36(8), 109602.

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Identification of iNKT Cell Subsets

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The following reagents were used to stain 2x10⁶ PBMCs for ex vivo iNKT analysis: PE-conjugated CD1d-αGC tetramers, FITC anti-Vα24 (Clone C15, Beckman Coulter, High Wycombe, UK) and APC anti-CD3 (Clone SP34-2, BD Biosciences, Oxford, UK). In vitro αGC expanded iNKT cells were stained with the following reagents: PE-Cy7 anti-CD8 (Clone SK1, Biolegend, London, UK), PerCP-Cy5.5 anti-CD4 (Clone OKT4, Biolegend, London, UK), PE-conjugated CD1d-αGC tetramers, FITC anti-Vα24 and APC anti-CD3 to identify iNKT subsets. Propidium iodide (Sigma-Aldrich, Gillingham, UK) was added to identify live cells, before acquisition on either a FACSCalibur or a FACSAria II (BD Biosciences, Oxford, UK). Analysis was performed using Cell Quest (version 0.3.bfab, BD Biosciences, San Jose, US) or FloJo (version 9.7.6, Treestar, Ashland, OR, US) software; and cells were gated on live, CD3⁺ lymphocytes.

Chancellor A., White A., Tocheva A.S., Fenn J.R., Dennis M., Tezera L., Singhania A., Elliott T., Tebruegge M., Elkington P., Gadola S., Sharpe S, & Mansour S. (2017). Quantitative and qualitative iNKT repertoire associations with disease susceptibility and outcome in macaque tuberculosis infection. Tuberculosis (Edinburgh, Scotland), 105, 86-95.

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Sorting and Expansion of Th1, Th1/Th17, and Th17 Cells from RRMS Patients

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