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Protocol For CD154-Enrichment 150113
Protocol For CD154-Enrichment 150113
Aim:
Ex vivo enrichment of CD154+ expressing T cells following stimulation with different antigens
in combination with intracellular cytokine staining
Materials:
RPMI-1640 medium
human AB-Serum
Brefeldin A
CD40 functional grade pur
CD28 functional grade pur
anti human CD154-Biotin
anti-Biotin Microbeads (CD154 Microbead Kit contains both components)
PEB-Puffer (1xPBS, 2 mM EDTA, 0,5% BSA) (keep buffer cold (4-8°C)
MACS MS Columns
antigens
Inside Fix solution
Inside Perm solution
1) In vitro stimulation for induction of CD154+ expression
• sterile preparation of PBMCs from buffy coats or whole blood via Ficoll
(if possible take fresh blood; we get the buffy coats in the evening, than I make PBMCs, put
them overnight in the incubator and stimulate the cells in the next morning)
• incubate cells for 5 hours at 37°C, 5%CO2; add 1µg/ml BrefeldinA and incubate for
another 2hours
(it is important to keep this stimulation times to have enough time for CD154 to be up-
regulated!)
2) Magnetic labeling
• collect cells by pipetting up and down; rinse dish with cold buffer
• labeling for 108 total cells: (for 107 total cells scale down volumes accordingly; keep titer
1:10 for biotin and bead labeling)