Cd8 percp cy5

Manufactured by BioLegend

Sourced in United States

CD8-PerCP-Cy5.5 is a fluorescent-conjugated antibody that binds to the CD8 cell surface antigen. It is designed for use in flow cytometry applications to identify and quantify CD8-positive cells in a sample.

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Close spelling variants of this product

CD8-PerCP-Cy5.5 (clone 53-6.7) (2 citations) CD8-Percp-cy5.5 (clone SK1, (2 citations) Anti-CD8- PerCP-Cy5.5 (clone SK1, (2 citations) Anti-CD8-PerCP/Cy5.5 (27 citations) Anti-CD8-PerCP/Cy5.5 (clone 53–6.7) (3 citations) Anti-mouse CD8-PerCP-Cy5.5 (4 citations) PerCP-Cy5.5-anti-CD8 (4 citations) CD4 PE-Cy7 (SK3); CD8 PerCP-Cy5.5 (RPA-T8); CCR7 FITC (G043H7); CD45RA BV421 (HI100); HLA-DR PE (L243); CD38 BV605 (HIT2) (2 citations) PerCP/Cy5.5-CD8 (7 citations) PerCP/Cy5.5-conjugated anti-CD8 (6 citations)

54 protocols using cd8 percp cy5

Tetramer-based Identification and Sorting of B Cells

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B cells were eluted from PBMCs using a MACS Human B Cell isolation kit (Miltenyi Biotec). B cells were stained with rGP38 (IbAr10200) that had been tetramerized at 25 nM using Streptactin-PE (IBA Lifesciences) and Streptactin-APC (IBA Lifesciences). B cells were simultaneously stained with rGP38-Streptactin-PE and rGP38-Streptactin-APC tetramers for 1 hour on ice. Cells were washed twice in buffer (PBS, FBS, EDTA). Next, B cells were stained with a panel of antibodies. Donor 1 PBMCs were stained with a cocktail of anti-human CD3 PerCP-Cy5.5 (Biolegend), CD8 PerCP-Cy5.5 (Biolegend), CD14 PerCP-Cy5.5 (Invitrogen), CD16 PerCP-Cy5.5 (Biolegend), propidium iodide (PI) (Invitrogen), CD19 PE-Cy7 (Biolegend), CD27 BV510 (BD Biosciences), IgM BV711 (BD Biosciences), IgD BV421 (Biolegend), IgG BV605 (BD Biosciences), and IgA AF488 (Abcam). Donor 5 and 6 PBMCs were stained with a cocktail of anti-human CD3 PerCP-Cy5.5 (Biolegend), CD8 PerCP-Cy5.5 (Biolegend), CD14 PerCP-Cy5.5 (Invitrogen), CD16 PerCP-Cy5.5 (Biolegend), PI (Invitrogen), CD19 PE-Cy7 (Biolegend), CD20 PE-Cy7 (Biolegend), CD27 BV510 (BD Biosciences), IgM AF488 (Biolegend), and IgD BV421 (Biolegend). B cells were washed twice in buffer and run on a FACS Aria Fusion Cytometer (BD Biosciences). B cells were sorted into Super Script III reaction buffer (ThermoFisher Scientific) in 96-well Costar plates and frozen at −80 °C.

Shin O.S., Monticelli S.R., Hjorth C.K., Hornet V., Doyle M., Abelson D., Kuehne A.I., Wang A., Bakken R.R., Mishra A., Middlecamp M., Champney E., Stuart L., Maurer D.P., Li J., Berrigan J., Barajas J., Balinandi S., Lutwama J.J., Lobel L., Zeitlin L., Walker L.M., Dye J.M., Chandran K., Herbert A.S., Pauli N.T, & McLellan J.S. (2024). Crimean-Congo Hemorrhagic Fever Survivors Elicit Protective Non-Neutralizing Antibodies that Target 11 Overlapping Regions on Viral Glycoprotein GP38. bioRxiv.

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Quantifying Immune Cell Profiles in Intracranial Melanoma

Cited 3 times

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Flow cytometry was used to analyze immune cell populations (12 (link), 13 (link), 44 (link)) in a minimum of three samples in two independent animal studies. Dissected intracranial melanoma tumors were physically dissociated and filtered through a 70-μm cell filter. Single cells were labeled with primary antibody. Cells without primary antibody labeling were used as unlabeled negative controls; fluorescent beads (UltraComp eBeads^™, Invitrogen^™, Carlsbad, CA) were used as positive/calibration controls and to determine compensation between fluorescent channels. Forward- and side-scatter gating identified single cells and viable cell (Ghost Dye^™ Red 780 Viability Dye, 1:100; Tonbo Biosciences, San Diego, CA) exclusion identified live cells. Fluorescence minus one (FMO) methodology was used to determine gating. Flow cytometry was performed on an Attune NxT Flow Cytometer (Thermo Fisher Scientific^™ Inc., Waltham, MA), and compensation matrix computed and data analyzed using FlowJo version 9 software (Ashland, OR) following published flow cytometry guidelines (45 (link)).
Antibodies used were: CD4 (BV510, 1:400, clone RM4–5), CD8 (PerCP-cy5.5, 1:200, clone 53–6.7), FOXP3 (BV421, 1:100, clone MF-14), all acquired from BioLegend^® Inc. (San Diego, CA); and CD45 (PE-cy7, 1:200, clone 30-F11), CD3 (FITC, 1:200, clone 17A2), both acquired from Tonbo Biosciences.

Clark P.A., Sriramaneni R.N., Bates A.M., Jin W.J., Jagodinsky J.C., Hernandez R., Le T., Jeffery J.J., Marsh I.R., Grudzinski J.J., Aluicio-Sarduy E., Barnhart T.E., Anderson B.R., Chakravarty I., Arthur I.S., Kim K., Engle J.W., Bednarz B.P., Weichert J.P, & Morris Z.S. (2021). Low-Dose Radiation Potentiates the Propagation of Anti-Tumor Immunity against Melanoma Tumor in the Brain after In Situ Vaccination at a Tumor outside the Brain. Radiation research, 195(6), 522-540.

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Detecting Tim Expression in T-cells

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To detect the expression of Tims in various T-cell subpopulations, PBMCs were surface stained with the following anti-human mAbs: CD3 Pacific Blue, CD4 FITC, CD8 PerCP-Cy5.5, Tim-1 PE and Tim-3 BB515 (BioLegend, San Diego, CA, USA). Dead cells were excluded from analysis by staining with the Fixable Viability Stain 780 (BD, Franklin lakes, NJ, USA) and analysed using FlowJo Software X (Tree Star, Ashland, OR, USA). The gating strategy used for T-cell subsets is shown in Figure S1.

Liu Y., Chen Z., Xiao Y., Chen H, & Zhou Z. (2022). Altered expression of Tim family molecules and an imbalanced ratio of Tim-3 to Tim-1 expression in patients with type 1 diabetes. Frontiers in Endocrinology, 13, 937109.

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Immune Cell Analysis in Musculoskeletal Tissue

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To analyze immune cells in the musculoskeletal tissues at 3 and 7 dpi, ipsilateral feet were perfused extensively, skinned, disjointed from the tibia, and digested in RPM11640 medium supplemented with 10% FBS, 15 mM HEPES, 0.5 mg/ml of collagenase (Sigma, C0130) and 10 ug/ml of DNase I (Sigma, D5025) for 1 h at 37°C. Digested tissue was passed through a 70 μm strainer, and cells were separated by centrifugation and washed with PBS supplemented with 2% FBS and 2 mM EDTA. Cells were stained with a Fixable Viability Dye eFluor 506 (eBioscience, 65-0866-14) and incubated with the following antibodies for 1 h at 4°C: CD16/32 (Biolegend, 101301, 1:200) CD11b PE-Dazzle 594 (BioLegend, 101256, 1:200), Ly6G PerCP-Cy5.5 (Biolegend, 127616, 1:400), Ly6C Pacific Blue (BioLegend, 128014, 1:200), F4/80 APC (Thermo Fisher, 17-4801-82, 1:200), CD11c PE-Cy7 (BD Biosciences, 558079, 1:200), I-A/I-E (MHC class II) Alexa Fluor 700 (BioLegend, 107622, 1:200), NK-1.1 PE (BioLegend, 108708, 1:200), CD3 BUV737 (BD Biosciences, 564380, 1:200), CD8 PerCP-Cy5.5 (BioLegend, 100734, 1:200), CD4 BV786 (BioLegend, 100552, 1:200), B220 BV711 (BioLegend, 103255, 1:200). Cells were analyzed using a BD X20 Fortessa flow cytometer, and all data were processed using FlowJo software (FlowJo, LLC).

Zhang R., Earnest J.T., Kim A.S., Winkler E.S., Desai P., Adams L.J., Hu G., Bullock C., Gold B., Cherry S, & Diamond M.S. (2019). Expression of the Mxra8 Receptor Promotes Alphavirus Infection and Pathogenesis in Mice and Drosophila. Cell reports, 28(10), 2647-2658.e5.

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Evaluating Dexamethasone's Impact on CTL Viability

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Annexin V apoptosis assay was performed to evaluate the effect of dexamethasone on the viability of CTLs from Cas9 control and NR3C1 KO groups. CTLs from both groups were treated with 200 μM dexamethasone (Sigma) for 72 hours. Cells were then collected, washed with Annexin V buffer, and stained with Annexin V (V500; BD Biosciences) and live/dead viability dye (efluor 660; Invitrogen) in addition to CD3 APC Cy7 (Biolegend, Clone HIT3A), CD4 APC (E Biosciences, Clone SK3), and CD8 PerCP Cy5.5 (Biolegend, Clone SK1). The proportion of apoptotic (positive for Annexin V) and dead CTLs (positive for live/dead stain) was determined by flow cytometry.
To confirm the ability of SARS-CoV-2 CTLs to mediate cytotoxicity, autologous PBMCs were labeled with carboxyfluorescein succinimidyl ester (CFSE) per manufacturer’s recommendation and loaded with SARS-CoV-2 Spike (S) (peptide pool 1 or 2), Membrane (M) and Nucleocapsid (N) PepMix (JPT, Germany) [1μg /ml per peptide] overnight. The next day, the SARS-CoV-2 PepMix-loaded PBMCs were co-cultured with the expanded CTLs at 1:1 ratio for 16 hours followed by Annexin V staining as described above. The proportion of dead/apoptotic PBMCs (Live/Dead+ and Annexin V+) was determined by flow cyometry.

Basar R., Uprety N., Ensley E., Daher M., Klein K., Martinez F., Aung F., Shanley M., Hu B., Gokdemir E., Nunez Cortes A.K., Mendt M., Reyes Silva F., Acharya S., Laskowski T., Muniz-Feliciano L., Banerjee P.P., Li Y., Li S., Melo Garcia L., Lin P., Shaim H., Yates S.G., Marin D., Kaur I., Rao S., Mak D., Lin A., Miao Q., Dou J., Chen K., Champlin R.E., Shpall E.J, & Rezvani K. (2021). Generation of glucocorticoid-resistant SARS-CoV-2 T cells for adoptive cell therapy. Cell Reports, 36(3), 109432.

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Isolation and Characterization of NP105-113-Specific CD8+ T Cells

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NP_105–113-B*07:02-specific CD8⁺ T cells were stained with phycoerythrin (PE)-conjugated HLA-B7 NP_105–113 pentamer (ProImmune). Live/dead fixable Aqua dye (Invitrogen) was used to exclude nonviable cells from the analysis. Cells were washed and stained with the following surface antibodies: CD3-FITC (BD Biosciences), CD8-PercP-Cy5.5, CD14-BV510, CD19-BV510 and CD16-BV510 (BioLegend). After exclusion of nonviable/CD19⁺/CD14⁺/CD16⁺ cells, CD3⁺CD8⁺pentamer⁺ cells were sorted directly into 96-well PCR plates (Thermo Fisher Scientific) using a BD Fusion sorter or BD FACS Aria III (BD Biosciences) and stored at −80 °C for subsequent analysis.

Peng Y., Felce S.L., Dong D., Penkava F., Mentzer A.J., Yao X., Liu G., Yin Z., Chen J.L., Lu Y., Wellington D., Wing P.A., Dominey-Foy D.C., Jin C., Wang W., Hamid M.A., Fernandes R.A., Wang B., Fries A., Zhuang X., Ashley N., Rostron T., Waugh C., Sopp P., Hublitz P., Beveridge R., Tan T.K., Dold C., Kwok A.J., Rich-Griffin C., Dejnirattisa W., Liu C., Kurupati P., Nassiri I., Watson R.A., Tong O., Taylor C.A., Kumar Sharma P., Sun B., Curion F., Revale S., Garner L.C., Jansen K., Ferreira R.C., Attar M., Fry J.W., Russell R.A., Stauss H.J., James W., Townsend A., Ho L.P., Klenerman P., Mongkolsapaya J., Screaton G.R., Dendrou C., Sansom S.N., Bashford-Rogers R., Chain B., Smith G.L., McKeating J.A., Fairfax B.P., Bowness P., McMichael A.J., Ogg G., Knight J.C, & Dong T. (2021). An immunodominant NP105–113-B*07:02 cytotoxic T cell response controls viral replication and is associated with less severe COVID-19 disease. Nature Immunology, 23(1), 50-61.

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MHC Class I Tetramer Staining Protocol

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All MHC class I tetramers were made by the NIH Tetramer Core Facility. Up to five tetramer-peptide reagents with contrasting fluorescence were used in a given staining cocktail. The amount of each tetramer was titrated (0.1–1 μL) to obtain the optimal concentration for usage. Freshly isolated and 14-day cultured cells were washed with PBS, stained with tetramer cocktail in PBS + 2% FBS, and incubated at 4 °C for 30 min. An antibody cocktail comprised of CD8-PerCP/Cy5.5, CD45RA-BV510, CD62L-PE/Cy7, CD95-PE/Dazzle 594 (Biolegend) was added and samples incubated for an additional 30 min at 4 °C. Samples were then washed with FACS buffer and analyzed by flow cytometry (CytoFLEX, Beckman Coulter).

Choy C., Chen J., Li J., Gallagher D.T., Lu J., Wu D., Zou A., Hemani H., Baptiste B.A., Wichmann E., Yang Q., Ciffelo J., Yin R., McKelvy J., Melvin D., Wallace T., Dunn C., Nguyen C., Chia C.W., Fan J., Ruffolo J., Zukley L., Shi G., Amano T., An Y., Meirelles O., Wu W.W., Chou C.K., Shen R.F., Willis R.A., Ko M.S., Liu Y.T., De S., Pierce B.G., Ferrucci L., Egan J., Mariuzza R, & Weng N.P. (2023). SARS-CoV-2 infection establishes a stable and age-independent CD8+ T cell response against a dominant nucleocapsid epitope using restricted T cell receptors. Nature Communications, 14, 6725.

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Comparative Analysis of ERAP1 Haplotypes

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Cloning of ERAP1 haplotypes has been described (26 (link)). For expression of short antigenic peptides, forward and reverse oligonucleotides were annealed and ligated into pcDNA3.1D/V5-His-TOPO® vector using the Directional TOPO Expression kit (Invitrogen) as described (22 (link)).
To compare ERAP1 Hap2 and Hap10 activity, ERAP1^-/- WM793 clones were transfected with pcDNA-ERAP1 Hap2 or Hap10 (100 ng) at 60-80% cell density using FuGENE HD (Promega). 24 h after transfection, HLA expression was evaluated, or Vα3S1/Vβ13S1-TCR hybridoma cells were added and assessed for sGFP induction after 24 h of co-culture by flowcytometry. Parental or ERAP1^-/- HEK293T cells were seeded on 48 well plates and co-transfected with pRSV-HLA-C*06:02 (75 ng), plasmid encoded ADAMTSL5 peptides (75 ng) and either pcDNA-ERAP1 Hap2, Hap10, or pcDNA-vector (75 ng) using FuGENE HD. 24 h later, Vα3S1/Vβ13S1-TCR hybridoma cells were added. Staining with CD8-PerCPCy5.5 (Biolegend #344710) differentiated hybridoma cells from ERAP1^-/- HEK293T cells. Gating strategy to examine TCR hybridoma stimulation is given in Supplemental Fig. 2A.

Arakawa A., Reeves E., Vollmer S., Arakawa Y., He M., Galinski A., Stöhr J., Dornmair K., James E, & Prinz J.C. (2021). ERAP1 Controls the Autoimmune Response against Melanocytes in Psoriasis by Generating the Melanocyte Autoantigen and Regulating its Amount for HLA-C*06:02 Presentation. Journal of immunology (Baltimore, Md. : 1950), 207(9), 2235-2244.

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Multiparametric Analysis of Liver-Derived Immune Cells

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A nonparenchymal cell fraction from whole liver extracts was isolated upon collagenase and mechanical digestion followed by Percoll (GE Healthcare Life Sciences) gradient centrifugation as previously described [5 (link)]. In parallel, blood samples were collected in EDTA-containing tubes and treated with red blood cell lysis buffer (PharmLyse, BD Biosciences, Germany). Upon removal of red bodies and centrifugation, immune cells were incubated with fluorochrome-conjugated antibodies and characterized according to two different panels, a myeloid panel: CD45-BV510 (103138, BioLegend), 7AAD-PE-Cy5-YG (420404, BioLegend), CD11b-BV711 (101242, BioLegend), F4/80-APC (17-4801-82, eBiosciences), MHC2-Alexa700 (107622, BioLegend), CD11c-PE-Cy7 (25-0114-81, eBiosciences), and Ly6G-FITC (551460, BD Pharmingen) and a lymphoid panel: CD45-BV510 (103138, BioLegend), 7AAD-PE-Cy5-YG (420404, BioLegend), CD3-PE-Cy7 (25-0031-82, eBiosciences), CD4-FITC (11-0041-85, eBiosciences), CD8-PerCpCy5.5 (126610, BioLegend), and NK1.1-BV711 (108745, BioLegend). Labeled cells were then subjected to flow cytometry using a BD Canto II (BD Biosciences) and relative cell numbers were analyzed using FlowJo software (Tree Star).

Ramadori P., Drescher H., Erschfeld S., Fragoulis A., Kensler T.W., Wruck C.J., Cubero F.J., Trautwein C., Streetz K.L, & Kroy D.C. (2017). Genetic Nrf2 Overactivation Inhibits the Deleterious Effects Induced by Hepatocyte-Specific c-met Deletion during the Progression of NASH. Oxidative Medicine and Cellular Longevity, 2017, 3420286.

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Flow Cytometry Sample Preparation

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Cells were transferred from the 96 well plate to a PCR plate (Thermo Fisher) and centrifuged at 400 x g for 5 min at room temperature, making sure to aspirate the resulting supernatant in between each transfer. Once all cells were transferred to the new PCR plate, cells were centrifuged and resuspended in FBS Stain Buffer (BD Biosciences). FC Block Cocktail (Human TruStain FcX Fc Receptor Blocking Solution [Biolegend] and FBS Stain Buffer) was added to each sample and incubated for 10 min at 21 °C. Following the blocking incubation, Live Stain Cocktail, composed of Live/Dead-Aqua (1:100, Biolegend), CD8-PerCP-Cy5.5 (1:20, Biolegend), and CD14-PE-Dazzle594 (1:20, Biolegend) in FBS Stain Buffer, was added to each well, and the plate was incubated for a subsequent 30 min at 21 °C. Samples were washed twice via centrifugation and resuspension in 150 μL of FBS Stain Buffer, and finally resuspended in 60 μL of FBS Stain Buffer. 16% PFA was added to each sample for a final concentration of 1.6% PFA and incubated for 10 min at 21 °C to fix the cells. After fixation, cells were washed as before and resuspended in FBS Stain Buffer in preparation for intracellular staining.

Chen H., Schürch C.M., Noble K., Kim K., Krutzik P.O., O’Donnell E., Vander Tuig J., Nolan G.P, & McIlwain D.R. (2020). Functional comparison of PBMCs isolated by Cell Preparation Tubes (CPT) vs. Lymphoprep Tubes. BMC Immunology, 21, 15.

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