The assay was performed according to the standardized Mye-EUNITER protocol as described.30 (link) Briefly, allogenic to monocytes CD3 T cells were isolated from healthy donor PBMC by MACS (Milteny Biotec) and labeled with 10 µM cell proliferation dye eFluor 450 (eBioscience) at RT for 20 min followed by the co-incubation for 4 days with monocytes prestimulated for 24 hours with rHSP90a (2 µg/mL). Cells were co-cultured in 96-well round-bottom plates (Sarstedt) precoated for 2 hours with anti-CD3 (1 µg/mL, clone OKT-3, eBioscience) and anti-CD28 antibodies (2 µg/mL, clone CD28.2, Beckman Coulter). T cell proliferation was measured by assessing proliferation dye eFluor 450 dilution by FACS-Lyric (BD Biosciences) flow cytometer.
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Mye-EUNITER T Cell Proliferation Assay
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Corresponding Organization : German Cancer Research Center
Other organizations : University of Southern California
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Variable analysis
independent variables
- RHSP90a (2 µg/mL)
- T cell proliferation
- Allogenic to monocytes CD3 T cells isolated from healthy donor PBMC by MACS (Milteny Biotec)
- T cells labeled with 10 µM cell proliferation dye eFluor 450 (eBioscience) at RT for 20 min
- Monocytes prestimulated for 24 hours with rHSP90a (2 µg/mL)
- Cells co-cultured in 96-well round-bottom plates (Sarstedt) precoated for 2 hours with anti-CD3 (1 µg/mL, clone OKT-3, eBioscience) and anti-CD28 antibodies (2 µg/mL, clone CD28.2, Beckman Coulter)
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