Citrate synthase activity assay kit

CS activity was measured by a kit (“Citrate Synthase Activity Assay Kit”, ab119692; Abcam, Cambridge, UK) according to manufacturer’s protocol and normalized for protein. The cells were seeded and differentiated until day 16 in 10 cm-dishes.

Cells were washed twice with phosphate-buffered saline and incubated for 30 min on ice in lysis buffer (68 mM sucrose, 200 mM mannitol, 50 mM potassium chloride, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM EGTA, and 1 mM dithiothreitol with a protease inhibitor cocktail. The cells were then lysed using 45 passages through a 25G 5/8 needle and centrifuged at 1,500 g for 10 min. Cytosolic extracts were recovered after centrifugation at 13,000 g for 20 min. The pellet contained the mitochondria. The total protein concentration was determined using the BCA kit (Pierce, USA) according to the manufacturer’s instructions. The mitochondrial activity of complex I (ab109903, Abcam), complex II + III (b109905, Abcam), complex IV (ab109906, Abcam), and complex V (ab109907, Abcam) was determined using in vitro assays following the manufacturer’s procedures. The citrate synthase activity of mitochondrial extracts was measured using a Citrate Synthase Activity Assay Kit (ab119692, Abcam) according to the manufacturer’s instructions.

1.5 × 10⁴ NFs, BCC and SCC CAFs were seeded in 96 well-plates and assayed when a confluency of ~ 70–80% was reached. The activity of the citrate synthase enzyme, a proxy for cellular mitochondria content, was measured using the Citrate Synthase Activity Assay Kit (Abcam, UK) following manufacturer’s instructions. The colorimetric readings were acquired using a microplate Tecan instrument (Tecan, Infinite 200 PRO model, Switzerland) and normalized to a cell-free blank and to the amounts of harvested cells for each sample. Each condition was assayed in three biological replicates, and each sample was tested in three technical replicates.

Citrate synthase activity was determined using a Citrate Synthase Activity Assay Kit (#ab239712, Abcam). BMDMs were treated with IR-61 (10 μM) or vehicle control for 24 h and then incubated with ice cold CS assay buffer for 10 min. After centrifugation at 10,000g for 5 min, the supernatant was collected and mixed with reaction buffer and then absorbance (OD412 nm) was measured immediately in kinetic mode at 25 °C for 40 min. Protein concentration was determined for normalization and the citrate synthase activity was displayed as nmol min⁻¹ mg⁻¹ protein.

Citrate synthase activity was measured by Citrate Synthase Activity Assay Kit (Abcam, Cambridge, UK). Mitochondrial complex I–III activity was assay as described in Barrientos et al. (2009 (link)). All values were normalized for total protein amount.

Activity of citrate synthase in IPSC-derived NPCs was determined using the Citrate Synthase Activity Assay Kit (Abcam, cat. no. ab119692). Data were measured as a change in absorbance per min per μg of protein after measuring the exact input protein per well. The measurements were obtained by kinetics reading at 412 nm for 10 min with shaking every 30 s using an EnVision^® 2105 multimode plate reader.