Cd8 pe vio770

Manufactured by Miltenyi Biotec

Sourced in Germany

The CD8-PE-Vio770 is a fluorochrome-conjugated monoclonal antibody that binds to the CD8 protein expressed on the surface of T cells. This reagent can be used for the identification and enumeration of CD8+ T cells in flow cytometric analyses.

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Anti-CD8-PE-Vio770 CD8-PE

6 protocols using cd8 pe vio770

Multicolor Flow Cytometry of Whole Blood

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We used CD3-APC-Vio770, CD4-PE, CD8-PE-Vio770, and CD45RO from Miltenyi Biotec (Germany) and CD197-CCR7-FITC from BD Biosciences (Belgium) to stain the whole blood for 30 minutes. A 6-color BD FacsVerse instrument (BD Biosciences) was used to conduct the flow cytometry, and Flow Jo (version 10) was used to analyze the data. The gating strategies are shown in Figure S2.

Ngalamika O., Mukasine M.C., Kamanzi P., Kawimbe M., Mujajati A., Tso F.Y., Lidenge S.J, & Mumba C. (2022). SARS-CoV-2-Specific T Cell Immunity in HIV-Associated Kaposi Sarcoma Patients in Zambia. Journal of Immunology Research, 2022, 2114285.

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Characterization of CD45RC T cell subpopulations

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The following conjugated antibodies were used to characterize CD45RC T cell subpopulations: CD3-VioGreen (REA613), CD4-PerCP-Vio700 (REA623), CD8-PE-Vio770 (REA734), from Milteny Biotec, Bergisch-Gladbach, Germany; CD45RA-APC (HI100) from BD Biosciences, San Jose, CA, USA; and CD45RC-FITC (MT2) from IQ Product, Houston, TX, USA. Cell viability was systematically assessed (LIVE/DEAD Fixable Near-IR Dead Cell Stain kit; Fischer Scientific, Pittsburgh, PA, USA). Briefly, 10⁶ cells were incubated with the viability dye according to the manufacturer’s recommendations before incubation with the antibodies. Data were collected using a FACS-Canto II (BD Biosciences) cytometer and analyzed using the FlowJo software, Ashland, OR, USA. The expression of CD45RC is bimodal on CD4⁺ T cells, some cells expressing low levels of CD45RC (CD45RC^low), and others expressing high levels (CD45RC^high). On CD8⁺ T cells, expression of CD45RC is trimodal, the first fraction of cells expressing low levels (CD45RC^low), the second fraction expressing intermediate levels (CD45RC^Int), and the last fraction expressing high levels of CD45RC (CD45RC^high). Figure S1 illustrates the gating strategy.

Lemerle M., Garnier A.S., Planchais M., Brilland B., Delneste Y., Subra J.F., Blanchet O., Blanchard S., Croue A., Duveau A, & Augusto J.F. (2019). CD45RC Expression of Circulating CD8+ T Cells Predicts Acute Allograft Rejection: A Cohort Study of 128 Kidney Transplant Patients. Journal of Clinical Medicine, 8(8), 1147.

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Multiparametric Flow Cytometry Analysis

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The following mAbs were used for staining and flow cytometric measurements: CD3-VioGreen (BW264/56), CD4-allophycocyanin-Vio770 (VIT4), CD8-PE-Vio770 (BW135/80), CD14-peridinin chlorophyll protein (PerCP) (TÜK4), CD20-PerCP (LT20), CD27-FITC (M-T271), CD28-FITC (15E8), CD45RA-VioBlue (T6D11), CD45RO-FITC (UCHL1), CD62L-FITC (145/15), CD107a-PE (1D4B), CD127-FITC (MB15-18C9), CD137-allophycocyanin, CD137-PE (4B4-1), CD154-VioBlue (5C8), CD178-PE (NOK-1), CD197 (CCR7)-allophycocyanin, CD197 (CCR7)-PE (150503), CD279-allophycocyanin (PD1.3.1.3), anti-IL-2-PE-Vio770 (N7.48A), anti-IL-4-PE (7A3-3), anti-IL-17-FITC (CZ8-23G1), anti-IFN-γ-FITC, anti-IFN-γ-PE-Vio770 (45-15), anti-TNF-α-PE, and anti-TNF-α-VioBlue (cA2) (all Miltenyi Biotec).
Soluble biotinylated pHLA-A*0201 molecules loaded with WT1₃₇ (VLDFAPPGA), WT1₁₂₆ (RMFPNAPYL), WT1₁₈₇ (SLGEQQYSV), WT1₂₃₅ (CMTWNQMNL), or cytomegalovirus (CMV) pp65₄₉₅ (NLVPMVATV) were produced as described previously.14 (link) Tetramerization was achieved by binding to streptavidin-PE or streptavidin-allophycocyanin (both BioLegend, San Diego, CA). In each case, 2·5 × 10⁶ cells were stained with 1 µg/ml tetramer for 45 min at 4°C.
Data were acquired using a MACSQuant flow cytometer and analysed with MACSQuantify software (both Miltenyi Biotec).

Schmied S., Gostick E., Price D.A., Abken H., Assenmacher M, & Richter A. (2015). Analysis of the functional WT1-specific T-cell repertoire in healthy donors reveals a discrepancy between CD4+ and CD8+ memory formation. Immunology, 145(4), 558-569.

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Activation Markers and Cytokines in T Cells

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Whole blood was stimulated with 10 μg/mL phytohemagglutinin (PHA, Sigma-Aldrich) for 6 h and 24 h. After 6 h, activation markers on T cells were measured using CD69-APC (clone: REA824), CD71-FITC (clone: REA902), CD154-VioBlue (REA238) and CD25-PE (clone: 3G10), CD3-VioGreen (REA613), CD4-APC-Vio770 (REA623), and CD8-PE-Vio770 (REA734) antibodies and propidium iodide as viability dye (all Miltenyi Biotec, Bergisch-Gladbach, Germany) using a MACSQuant 16 analyzer (Miltenyi Biotec). After 24 h, culture supernatants were collected for cytokine analysis.

in ‘t Veld A.E., Grievink H.W., van der Plas J.L., Eveleens Maarse B.C., van Kraaij S.J., Woutman T.D., Schoonakker M., Klarenbeek N.B., de Kam M.L., Kamerling I.M., Jansen M.A, & Moerland M. (2023). Immunosuppression by hydroxychloroquine: mechanistic proof in in vitro experiments but limited systemic activity in a randomized placebo-controlled clinical pharmacology study. Immunologic Research, 71(4), 617-627.

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Isolation and Characterization of PBMCs

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Blood samples were collected in heparin anticoagulant tube, transferred them to the centrifuge tubes, added Ficoll into the tubes, isolated, and collected the peripheral blood mononuclear cell (PBMC). The cell suspension was labeled with the following monoclonal antibodies: anti-human CD4-FITC (Biolegend) and CD8-PE-Vio770 (Miltenyi). For surface marker staining like CD4 and CD8, cells were maintained in the dark at 4 °C for 30 min. While for intracellular staining of granzyme B, cells were fixed and permeabilized with the Foxp3-staining kit (eBioscience) according to the manufacturer’s guidelines. After permeabilization, cell suspension was incubated in the dark at 4 °C for 20 min with anti-Granzyme B-PE (Miltenyi).

Wang Q., Che N., Lu C., Sun X., Wang Y., Wang Q., Tan W., Zhou L., Zhang X., Xu D., Gu L, & Zhang M. (2021). Correlation of peripheral CD4+GranzB+CTLs with disease severity in patients with primary Sjögren’s syndrome. Arthritis Research & Therapy, 23, 257.

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Proliferation of CD45RC T Cells in ESRD Patients

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CD45RC T cells were sorted from freshly isolated PBMC of end-stage renal disease (ESRD) patients and age/sex matched healthy donors (HD) using a FACS-Aria cytometer, BD Bioscience, San Jose, CA, USA. Briefly, after a gradient centrifugation, 2 × 10⁷ PBMCs were stained using a Cell Trace Violet proliferation kit (Thermofischer, San Jose, CA, USA) for proliferation assessment and then stained using CD4-BV421 (L3T4, BD Biosciences), CD8-PE-Vio770 (REA734, Miltenyi Biotec), and CD45RC-FITC (MT2, IQ Product). CD45RC^high and CD45RC^low subpopulations were sorted among CD4⁺ and CD8⁺ T cells. Purity was always routinely above 95%. Then, 5 × 10⁴ T cells were cultured at 37 °C in RPMI 1640 medium (containing 8% fetal calf serum) in 96-well round-bottomed microplates (Becton Dickinson, Franklin Lakes, NJ, USA), with or without a 1 µg/mL plate-bound anti-CD3 (Beckman-Coulter, Brea, CA, USA) and 0.5 µg/mL soluble anti-CD28 (Beckman-Coulter). After 72 h of culture, cells were harvested and proliferation was assessed using flow cytometry (FACS-Canto II; BD Biosciences).

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