Cd4 krome orange

Manufactured by Beckman Coulter

Sourced in Canada

The CD4-Krome Orange is a lab equipment product designed to detect and quantify CD4+ T cells. It is a fluorescently-labeled antibody that binds to the CD4 surface receptor on T cells, allowing for their identification and enumeration through flow cytometry analysis.

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Krome Orange (3 citations)

3 protocols using cd4 krome orange

Multiparametric T Cell Phenotyping

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The following antibodies were used in this study for T cell phenotyping: CD3-PerCP (clone SK7/Cat# 347344), CD25-APC AF700, CD4-Krome Orange (13B8.2/A96417), CD8-Pacific Blue (B9.11/A82791), CD3-APC (Beckman Coulter Inc. Brea, CA), Rat Anti-Mouse IgG1-APC (X56/550874) (BD Biosciences, San Jose, CA). PD-1-Percp Cy7 and TIM3-APC (BD Biosciences, San Jose, CA) were used as markers of T cell exhaustion. MUC1 antigen expression by tumor cells was measured using anti-MUC1, (Santa Cruz Biotechnology. Inc., Dallas, TX). CAR molecules were detected using Goat anti-human F(ab’)2 antibody conjugated with AlexaFluor647 (109–606-097) (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Cells were stained with saturating amounts of antibody (~5uL) for 20 min at 4 °C, washed (PBS, Sigma-Alrich, St. Louis, MO), and then acquired on Gallios™ Flow Cytometer (Beckman Coulter Inc., Brea, CA). Analysis was performed using Kaluza® Flow Analysis Software (Beckman Coulter Inc.).

Bajgain P., Tawinwung S., D’Elia L., Sukumaran S., Watanabe N., Hoyos V., Lulla P., Brenner M.K., Leen A.M, & Vera J.F. (2018). CAR T cell therapy for breast cancer: harnessing the tumor milieu to drive T cell activation. Journal for Immunotherapy of Cancer, 6, 34.

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T Cell Co-culture Assay with CAPAN-1

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T cells were co-cultured with 0.5×10⁶ CAPAN-1 PSCA/TGFβ/IL4 producing cell line at the specified effector:target ratio in 4 mL complete media in a 6-well plate. The cells were harvested every 3 days, labeled with CD3 APC, CD4 Krome Orange and CD8 Pacific blue antibodies (Beckman Coulter) and quantified by flow cytometer using CountBrightTM Absolute Counting Beads (approximately 0.2×10⁵ beads/20 μL added to each condition) (Invitrogen, Eugene, OR) and 5 μL of 7-AAD (BD Biosciences) to exclude dead cells. Total tumor and T cell numbers were back calculated from the viable cell numbers obtained by terminating acquisition at 2,000 beads.

Sukumaran S., Watanabe N., Bajgain P., Raja K., Mohammed S., Fisher W.E., Brenner M.K., Leen A.M, & Vera J.F. (2018). Enhancing the potency and specificity of engineered T cells for cancer treatment. Cancer discovery, 8(8), 972-987.

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Multiparametric Flow Cytometry of CSF and Blood

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CSF samples were collected in polypropylene tubes and were processed within 20 min. Cells were obtained from EDTA blood by erythrocyte lysis using VersaLyse buffer (Beckman Coulter, Krefeld, Germany) following the manufacturer's instructions. Cells were obtained from CSF by centrifugation (15 min, 290g, 4°C) and incubation in VersaLyse buffer. Cells were stained for 30 min at 4°C using the following fluorochrome-conjugated antibodies: CD14-FITC, CD138-PE, HLA-DR-ECD, CD3-PC5.5, CD56-PC7, CD4-APC, CD19-APCAlexafluor700, CD16-APCAlexafluor750, CD8-PacificBlue, and CD45-KromeOrange (all Beckman Coulter). T-cell subpopulations were further analyzed using the following fluorochrome-conjugated antibodies: CD45RA-FITC, CD27-PE, CD3-ECD, CCR7-PC5.5, CD25-PC7, CD56-APC, CD127-APCAlexafluor700, CD62L-APCAlexafluor750 or PD1-APCAlexafluor750, CD8-PacificBlue, and CD4-KromeOrange (obtained from Beckman Coulter or Ebioscience, Frankfurt, Germany). After washing, all samples were analyzed using the Navios™ flow cytometer (Beckman Coulter, Germany). The gating strategy to determine HLA-DR expression on CD4+ and CD8+ T cells and CD4+ and CD8+ T-cell subpopulations are described in Figures S1 and S2.

Grauer O.M., Reichelt D., Grüneberg U., Lohmann H., Schneider-Hohendorf T., Schulte-Mecklenbeck A., Gross C.C., Meuth S.G., Wiendl H, & Husstedt I.W. (2015). Neurocognitive decline in HIV patients is associated with ongoing T-cell activation in the cerebrospinal fluid. Annals of Clinical and Translational Neurology, 2(9), 906-919.

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