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HDAC activity assay

ML Maik Luu
KW Katharina Weigand
FW Fatana Wedi
CB Carina Breidenbend
HL Hanna Leister
SP Sabine Pautz
TA Till Adhikary
AV Alexander Visekruna
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Fluorometric HDAC activity was measured according to the manufacturer’s protocol (Bachem). Briefly, CD8+ T cells were cultured under CTL-inducing conditions for three days. Subsequently, CTL-derived cell lysates were incubated with various SCFAs (5 mM) or with TSA (500 nM) for 15 minutes. In some experiments, 25 mM acetate and 1 mM butyrate were used for HDAC assay. To measure the inhibitory activity of SCFAs on HDACs, the cell lysates were treated with fluorogenic substrate Ac-Arg-Gly-Lys-AMC (Bachem) for next 30 minutes. HDAC-inhibitory activity of SCFAs was calculated relative to the enzymatic activity of untreated CTLs (this value was arbitrarily set to 100%). The measurement of fluorescence (free AMC) was carried out at the FLUOstar Omega device (BMG Labtech).

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