Mx3005p

Core biopsies were taken from each kidney (n = 10) in situ (baseline) and after 6 h of perfusion. Biopsies were stored in RNA later solution (Invitrogen RNA later™ Soln.) for fixing. The tissue was lysed (Precellys Lysing Kit MK28-R) and RNA was extracted (Invitrogen PureLink™ RNA Mini Kit & Invitrogen TURBO DNA-free™ Kit). The RNA was used to generate the cDNAs for each sample by reverse transcription (Applied Biosystems High Capacity cDNA Reverse Transcription Kit). Primers for ICAM-1, IL-1β, IL-6, IL-8, HMGB-1, TLR4 and RPL4 were designed using NCBI Primer Blast (Appendix: Table 2). Relative qPCR (Stratagene Mx3005P) was then performed using SYBR Green (Applied Biosystems Power SYBR^® Green PCR Master Mix) on baseline and 6 h perfusion samples for all kidneys and all genes. Cycling conditions are shown in Appendix: Table 3. Samples were assayed in triplicate and Ct values were averaged for each gene. The housekeeping gene (RPL4) Ct value was subtracted from the average Ct value of other genes within that sample (ΔCt). Since the kidneys were paired, the control ΔCt value was subtracted from the Cytosorb adsorber ΔCt value (ΔΔCt). The relative fold change for each gene between treatment and control was then calculated using 2^−ΔΔCt. The same analysis was performed comparing the relative expression difference within each kidney between baseline and 6 h.

RNA was extracted from 200 μL fecal suspension using the BioRobot Universal system with the QIAamp One-For-All Nucleic Acid Kit (Qiagen, Hilden, Germany) in accordance with the manufacturer’s instructions. Influenza A virus was screened using qPCR assay that targeted the influenza Matrix gene. Detection was performed using a Stratagene Mx3005P thermocycler with an AgPath RT-PCR Kit (Applied Biosystems, Foster City, CA, USA) using 5 μL eluate in 25 μL total volume. Each run included 2 negative and 2 positive control samples along with 92 samples. The positive samples detected by qPCR were inoculated into the allantoic cavity of 9-day-old specific pathogen free (SPF) embryonated chicken eggs (ECEs). The ECEs were incubated at 37°C for 48 h and then chilled at 4°C for 6–8 h. Allantoic fluids were harvested and hemagglutination assays with 0.5% turkey red blood cells confirmed the presence of viruses.

RNA was isolated using 1 ml of TRIzol (Sigma-Aldrich) according to the manufacturer’s instructions. RNA was quantified using a NanoDrop (NanoDrop Technologies), and 1 μg of RNA was used for cDNA synthesis using Moloney Murine Leukemia Virus reverse transcriptase (Promega). The cDNA was analyzed by quantitative PCR performed using probe-based gene expression assays (IDT or Themo Fisher) in a Stratagene MX3005P or Applied Biosystems Quant Studio 3. Reactions were carried out in 20 μL and analyzed using the ΔΔCt method.