54 Chapter 2 Amino acid measurements Amino acid profiles were measured by GC/MS (Agilent 9575C series GC/MSD, Agilent Technologies, Amstelveen, The Netherlands) as previously described70. Albumin measurement Supernatant from the hepatic organoid cultures was collected every 24 hours starting from day 12 of the differentiation until day 16 and was stored at -20°C. The concentration of albumin in the supernatant was measured using the mouse albumin ELISA kit (Abcam, Cambridge, UK) according to the manufacturer’s instructions in the Synergy H4 Hybrid Microplate Reader (BioTek Instruments Inc., VT, USA). RNA isolation, reverse transcription and quantitative realtime qPCR Total RNA was extracted from organoids using the commercially available RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA quality and quantity was determined with NanoDrop (NanoDrop Technologies, Wilmington, DE, USA). Reverse transcription was completed with the M-MLV Reverse Transcriptase (200 U/µL) (invitrogen) according to the manufacturer’s instructions. qPCR reactions were performed using FastStart Universal SYBR Green Master (Rox) (Sigma Aldrich) in 384 well format in duplicate with 20 ng total cDNA per well in a QuantStudio 7 Flex (Thermo Fisher Scientific). All primers sequences (Integrated DNA technologies Inc., Coralville, IA, USA) are listed in Supplementary Table 2. Peptidylprolyl isomerase A (cycophilin A)(PPIA) served as endogenous control, as expression was stable between experimental groups and was used for normalization. Relative expression was calculated using the ΔΔC(t) method relative to the control organoids. Triglyceride measurement Organoids were collected in 1X TBS in MiliQ water. Fat was extracted using chloroform-methanol in a 2:1 ratio. Quantitative determination of hepatic triglycerides was done with the DiaSys Triglyceride FS kit (Holzheim, Germany, Cat #157109910971). Results were normalized to protein content.
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