Rna quick rna micro prep kit lysis buffer

As we wanted to activate CD14-dependent signalling, a low dose of LPS was necessary as it is known that higher concentrations of LPS result in CD14-independent TLR4 signalling [19 (link), 20 (link)]. In previous experiments using the mouse epithelial cell line CMT93, we tested different LPS concentrations and time points [11 (link)]. In our hands, 0.1 μg/ml and 6 hours were the best concentration and incubation time to measure CD14-dependent cytokine induction. After 10 days of growth, colonic organoids were stimulated with LPS (Sigma-Aldrich, St. Louis, USA) as described previously [11 (link)]. In brief, growth media was removed and replaced with 500 μl fresh growth media without Cellshield and containing either 0.1 μg/ml LPS or no LPS as the control. After 6 hours of incubation at 37°C and 5% CO₂, media was removed and the Matrigel® was dissolved in ice-cold DPBS. Two to 6 wells were pooled, and cells were centrifuged, resuspended in RNA Quick-RNA™ Micro Prep Kit Lysis Buffer (Zymo Research, Irvine, USA), and stored at -80°C for later RNA isolation.

The organoids used to assess the effects of ZellShield® on organoid kinetics were isolated and cultivated as mentioned above (Sections 2.2 and 2.3) over 3 weeks in the presence of ZellShield® until passage 3. The organoids from passage 3 were then cultivated for 10 days in the presence of ZellShield®, and the medium was changed every 3 days, with the last change occurring one day prior to the experiment. On day 10, the old organoid growth medium was replaced by fresh organoid growth medium with or without ZellShield®, and the organoids were cultured for 1, 2, 4, 6, and 12 hours at 37°C in the presence of 5% CO₂. After incubation, the supernatant was removed and stored at -20°C. The plate was immediately placed on ice, the Matrigel® was dissolved, and the organoid structures were disrupted by thorough pipetting after the addition of ice-cold DPBS. The suspension was centrifuged, and the pellet was resuspended in RNA Quick-RNA™ Micro Prep Kit Lysis Buffer (Zymo Research, Irvine, CA, USA) and stored at -80°C until further processing for quantitative real-time PCR (qPCR) analysis. Two wells of each condition were pooled to obtain the supernatant and lysed samples. The whole experimental setup from isolation to sample collection was repeated in five independent experiments.