CAR Jurkat cell lines, CHO-X, CHO–EpCAM and MCF-7 cells were washed once in culture medium and adjusted to a density of 1 × 106 (link) cells/ml. CAR Jurkat cells were co-incubated with each target cell line at a ratio of 1:1 (200 μl/well, 96 round-bottomed plate) for 4 h at 37 °C. The cells were washed twice with MACS buffer and stained with anti-human CD69 FITC (BioLegend, catalog no. 310904) for 25 min at 4 °C. The cells were then washed three times with MACS buffer, followed by analysis of CD69 expression on an AttuneTM NxT flow cytometer (ThermoFisher Scientific) with accompanying software.
Anti human cd69 fitc
Manufactured by BioLegend
Anti-human CD69 FITC is a fluorescently-labeled antibody that binds to the CD69 protein expressed on the surface of activated T cells, B cells, and natural killer cells. It can be used for the flow cytometric detection and analysis of activated immune cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Human ifn γ kit, by PerkinElmer (1 mentions)
Anti human cd25 apc, by BD (1 mentions)
Anti cd28 antibody, by BD (1 mentions)
Easysep human cd4 t cell isolation kit, by STEMCELL (1 mentions)
Anti cd3, by BD (1 mentions)
Facscanto 2, by BD (1 mentions)
Facsdiva, by BD (1 mentions)
Percp cy5.5 anti human cd3, by BD (1 mentions)
Human serum type ab, by Merck Group (1 mentions)
Rpmi 1640 medium, by Merck Group (1 mentions)
4 protocols using anti human cd69 fitc
1
CAR T-cell Activation Assay
2
CD4+ T Cell Activation and Cytokine Profiling
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Human CD4+ T cells were isolated from PBMCs using EasySep™ Human CD4+ T Cell Isolation Kit (STEMCELL, 19052). 96-well flat-bottom plates were coated with 0.25 μg/mL anti-CD3 (BD, 555329) and incubated with varying concentration anti-GITR antibody at 37°C for 2 h or overnight at 4°C. On the following day, the plate was washed, and 2 × 104 human CD4+ T cells and 2 μg/mL anti-CD28 antibody (BD, 555725) were added. After 3 days, supernatant IL-2 and IFN-γ were measured using Human IL-2 kit (Cisbio, 62HIL02PEG) and Human IFN-γ kit (Cisbio, 62HIFNGPEG), respectively. Cell surface activation markers were analyzed by flow cytometry using anti-human CD69 FITC (Biolegend, 310904) and anti-human CD25 APC (BD, 555434).
3
Multicolor Flow Cytometry Immunophenotyping
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Cells were stained with PE-Cy7 mouse anti-human CD45RO (#560608; BioLegend), FITC mouse anti-human CD45RA (#130-092-247; Miltenyi Biotec), and FITC anti-human CD69 (#310904; BioLegend) antibodies. Flow cytometry was performed on the BD FACS Canto II using the BD FACSDIVA.
4
Fluzone Vaccine-Induced DC Activation Assay
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PBMCs were isolated eight months after the Fluzone vaccination. DCs were prepared from the PBMCs as described before (26 (link)). DCs were dissociated by using trypsin-EDTA and washed. Transfected Jurkat cells were washed, and then 4 × 104 Jurkat cells were mixed with or without 4 × 104 DCs in a total volume of 200-μl RPMI 1640 medium containing 5% human serum type AB (no. H4522; Sigma-Aldrich) and with or without 2.5-μl Fluzone Vaccine 2011-2012 (no. NR-36747; BEI resources) and incubated for 24 hours in a 96-well plate. The cells were stained at 4°C for 20 min in phosphate-buffered saline/0.2% bovine serum albumin/2 mM EDTA with FITC anti-human CD69 (no. 310904; BioLegend), PE anti-mouse TCRβ chain (no. 109207; BioLegend), and PerCP-Cy 5.5 anti-human CD3 (no. 552852; BD Biosciences). Cells were washed and analyzed with BD FACSAria and FlowJo v10 software.
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