Ab46052

Control, stress and DM preconditioned culture media was collected and stored at −40°C before use. NF-κB (P65) transcription factor ELISA (Abcam cat # ab133112) was performed on nuclear extracts instead of media. Collected media was used in triplicate. Cytokine concentrations were measured to further confirm the protective potential of DM extract. Collected media was subjected to ELISA for IL1β (Abcam cat # ab46052), TNF-α (Abcam cat # ab181421), IKKα and IKKβ (Cytoglow cat # CB5358) according to manufacturer’s protocol.

PBMCs, isolated as described in the section PBMC isolation, were used for monocyte purification using negative selection for CD14++CD16- monocytes (Miltenyi, 130-117-337) according to the manufacturer’s protocol. Before plating, purity of the samples was evaluated on flowcytometry by CD14 staining (>80% of total cells). Monocytes were counted, resuspended in macrophage serum free medium (Gibco, 12065-074) and seeded in a 96 well plate at a density of 1x10⁶ cells/ml in 100 µL with GM-CSF 10 ng/ml (Biolegend, 572902). Medium was changed every 3 days and macrophages were harvested at day 7. Macrophages were primed with lipopolysaccharide (LPS, 1 µg/ml; Sigma) for 6 h to induce the expression of IL1B, and thereafter activation of the NLRP3 inflammasome was induced with adenosine triphosphate (ATP, 5 mM, 45 min). Secretion of the mature IL-1β was measured by ELISA (Abcam, ab46052) according to manufacturer´s recommendations. Cells were further used for RNA extraction as described in the section isolation of mRNA and generation of cDNA.

ELISA tests were used according to the instructions of the manufacturer to determine the levels of the cytokines secreted in the cell culture medium. The following ELISA kits were used: IL-1β, sensitivity—6.5 pg/mL (Abcam, ab46052, Cambridge, UK); IL-6, sensitivity—0.7 pg/mL (Quantikine, R&D, D6050, Milan, Italy). The results of the ELISA tests were assessed using an Opsys MR microtiter plate reader (Dynex Technologies, Chantilly, VA, USA).

To generate three-tissue platforms, microreactors with liver organoids and cardiac organoids were connected in series with lung modules as shown in the schematic in Fig. 1a. Briefly, the pump drew media from the fluid reservoir and flowed it via the central routing breadboard to the liver, then the heart, followed by the lung, after which it circulated back to the reservoir. Flow rates of 10 μL/min and α-MEM (Hyclone, Logan, UT) with 10% FBS (Hyclone), 1% Pen/Strep (Thermo Fisher), and 1% L-glutamine (Thermo Fisher) were used in all of the subsequent fluidic platform studies. As additional controls, systems with only cardiac microreactors were prepared and subjected to the conditions described below.
After initiation of the three-tissue platforms, on day 3 bleomycin (10 μg/mL, Sigma) was added to half of the systems (n = 4), while the remaining half did not receive any drug treatment. Media aliquots were removed from the reservoir on days 1, 3, 5, 7, and 9. On day 9, cardiac organoids were assessed for beat rate analysis as described above, after which all organoids and constructs were assessed for viability by LIVE/DEAD and macro-confocal imaging as described above. Levels of IL-8 and IL-1β at each time point were assessed by ELISA (IL-8: ab46032, Abcam; IL-1β: ab46052, Abcam).

Plasma level of human interleukin-1 beta (IL-1β) was measured according to the manufacturer’s protocols (Abcam Inc, Cambridge, MA, USA, catalog # Ab 46052). A 100-μL of standard, control and tested samples in duplicate were added to the wells. Biotinylated anti-IL-1β was then added, and the plate was incubated for 3 h at room temperature. After 3 washings, 100-μL of streptavidin- HRP solution was added into all wells and incubated for 30 min. Next, plate was washed 3 times and each well was incubated with 100 μL of TMB in the dark for 15 min at room temperature followed by 100-μL of acidic stop solution. The absorbance at 450 nm was recorded using the microplate reader.

The blood samples of all patients and healthy controls were collected within 24 h after admission. The serum levels of HMGB1, I-FABP, and inflammatory factors CRP, IL-1β, IL-6 and TNF-α were determined by enzyme-linked immunosorbent assay (ELISA) using commercial ELISA kits (HMGB1 LifeSpan Biosciences LS-F26519, I-FABP Abcam ab193700, CRP Abcam ab181416, IL-1β Abcam ab46052, IL-6 Abcam ab178013, and TNF-α Abcam, ab181421), according to manufacturer’s instructions.

Cells were cultured (1 × 10⁶ cells/mL) for 72 h in 20 mM glucose containing the indicated concentrations of NTS or MSM. IL-1β, IL-6, and TNF-α were measured in the supernatants of cultured cells using a human ELISA kit (abcam; IL-1β: ab46052, IL-6: ab178013, and TNF-α: ab181421) according to the manufacturer’s instructions.