Dendritic Cell-T Cell Co-culture Assay

BMDCs were cultivated from fresh or immortalized (Drobek et al., 2018 (link), Ruedl et al., 2008 (link)) hematopoietic stem cells. The dendritic cells were pulsed with indicated concentration of indicated peptides and mixed with isolated T cells or thymocytes in ratio 1:1 or 1:2 and co-cultured in RPMI (Sigma Aldrich) containing 10% FBS (GIBCO), 100 U/ml penicillin (BB Pharma), 100 μg/ml streptomycin (Sigma Aldrich), 40 μg/ml gentamicin (Sandoz) for indicated period of time (pERK1/2 analysis) or over-night (CD25 and CD69 analysis). The EC₅₀ values for the CD69 upregulation assay were calculated using non-liner fit (log(agonist) versus response–Variable slope (four parameters)) in GraphPad Prism 5. The cell numbers were counted using Z2 Coulter Counter Analyzer (Beckman Coulter).

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Horkova V., Drobek A., Mueller D., Gubser C., Niederlova V., Wyss L., King C.G., Zehn D, & Stepanek O. (2019). Dynamics of the Coreceptor-LCK Interactions during T Cell Development Shape the Self-Reactivity of Peripheral CD4 and CD8 T Cells. Cell Reports, 30(5), 1504-1514.e7.

Publication 2019

RPMI FBS streptomycin gentamicin Z2 Coulter Counter Analyzer

Assay Cd25 Dendritic cells Gentamicin Hematopoietic stem cells Liner Penicillin Peptides Prism Streptomycin Thymocytes Upregulation

Corresponding Organization : Czech Academy of Sciences, Institute of Molecular Genetics

Other organizations : University of Basel, University Hospital of Basel, Peter Doherty Institute, University of Melbourne, Ludwig-Maximilians-Universität München, Technical University of Munich

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Variable analysis

independent variables

Fresh or immortalized hematopoietic stem cells
Indicated concentration of indicated peptides

dependent variables

Phosphorylation of ERK1/2 (pERK1/2 analysis)
Upregulation of CD25 and CD69 (CD25 and CD69 analysis)
Cell numbers (counted using Z2 Coulter Counter Analyzer)

control variables

Ratio of dendritic cells to T cells or thymocytes (1:1 or 1:2)
Culture medium (RPMI containing 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 40 μg/ml gentamicin)
Incubation time (indicated period of time for pERK1/2 analysis, over-night for CD25 and CD69 analysis)

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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