Acetyl coenzyme a assay kit

50 ml of yeast cell culture were harvested and immediately processed for acetyl-CoA extraction as previously described [90 (link)]. Acetyl-CoA level was measured using an Acetyl-Coenzyme A assay kit (Sigma-Aldrich; cat. # MAK039) according to the manufacturer’s instructions. A SynergyH1 Multi-Mode Plate Reader (BioTek) was used to measure the fluorometric product (λ_ex5 = 535/λ_em = 587) of each assay reaction.

Acetyl-CoA levels were measured using Acetyl-Coenzyme A Assay Kit, (#MAK039, Sigma). Acetyl-CoA concentration was determined based on fluorometric (ex = 535/em = 587 nm) product by coupled enzyme assay proportional to the metabolite present. The samples were prepared from ~ 0.1 million cells for experimental set. Sample was deproteinization using 25% (wt/vol) TCA final concentration followed by neutralization using 5 M KOH solution till pH 7–8 was achieved. Further, samples were processed for acetyl-CoA measurement as per manufacturer's instructions.

Naïve CD4 T cells were differentiated under hypoxia for 4 days before cultured in fresh Click’s medium for another 24h with or without 20mM sodium acetate. Cells were lysed with M-PER buffer for 10 minutes on ice. Cell lysates were spun down at 13000 rpm for 10min at 4 °C. Supernatant was deproteinized using 4M perchloric acid and neutralized by 1N potassium hydroxide. Then deproteinized lysates were used for acetyl-coA measurement using Acetyl-Coenzyme A Assay Kit (Sigma, #MAK039) following the manufacturer’s instructions. Fluorescence intensity was measured at Ex/Em=535/587nM. Acetyl-coA levels were normalized to cell number.

Cells were sampled during the course of lycopene shake-flask fermentation for acetyl-CoA assay. Acetyl-CoA was extracted as previously described [22 (link)] and analyzed by the acetyl-CoenzymeA Assay Kit (Sigma-Aldrich). The acetyl-CoA concentration was normalized by dry cell weight.

Acetyl‐coenzyme A was measured according to the manufacturer's protocol (acetyl‐coenzyme A assay kit, Sigma). All samples and standards were analysed in duplicate. Ten microlitres of each sample was added to duplicate wells of a 96‐well plate. Samples were brought to a final volume of 50 μL with acetyl‐CoA assay buffer. A blank sample for each sample was prepared by omitting the Conversion Enzyme in the Reaction Mix. Ten microlitres of acetyl‐CoA quencher was added to each sample, standard and sample blank well to correct for the background signal created by free coenzyme A and succinyl‐CoA. Plates were incubated at room temperature for 5 minutes. Two microlitres of Quench Remover was added to each well, mixed well and incubated for an additional 5 minutes. The plate was mixed well using a horizontal shaker or by pipetting, and the reaction proceeded for 10 minutes at 37℃ in the dark. The fluorescence intensity was subsequently measured (λex = 535/λem = 587 nm).