Qiashredder homogenizer

PBL of a healthy horse were dissolved in lysis buffer (9 M Urea, 2 M Thiourea, 1% Dithioerythritol, 4% CHAPS, 2.5 µM EDTA) and processed using QIAshredder homogenizers (Qiagen, Hilden, Germany) for depletion of DNA precipitates. Protein content of cell lysates was determined by Bradford protein assay (Sigma-Aldrich, Deisenhofen, Germany). One mg of lysate was loaded on 24 cm pH 3-11 NL IPG strips (GE Healthcare, Freiburg, Germany) by overnight reswelling and subjected to isoelectric focusing (IEF) on a 2117 Multiphor II Electrophoresis Unit with an Amersham Electrophoresis Power Supply EPS 3501 XL (Step 1: 2 h/50 V/2 mA/5 W, Step2: 11 h/600 V/10 mA/10 W, Step 3: 4 h/2000 V/10 mA/10 W, Step 4: 12 h/3000 V/10 mA/10 W). Strips were then equilibrated in 1% Dithiothreitol followed by 4.8% Iodoacetamide for 10 minutes each and sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) was subsequently performed in an Ettan DALT Six Electrophoresis Unit 230 (GE Healthcare) with an Amersham Electrophoresis Power Supply EPS 3501 XL (Step 1: 45 min/600 V/150 mA/9 W, Step 2: 1 h/850 V/300 mA/60 W, Step 3: 8 h/1000 V/400 mA/90 W). Resulting gels were colloidal coomassie stained and scanned on a transmission scanner. As many spots as possible were cut from gels and processed for mass spectrometry.

Total RNA was extracted from cells using RNeasy Protect Cell Mini Kit (Qiagen) with QIAshredder homogenizers (Qiagen). RNA was treated with DNase I (PrimerDesign or Invitrogen) according to manufacturer’s instruction, samples’ concentration was adjusted to 50 ng/µl and verified on Qubit Fluorometer (Invitrogen) using Qubit RNA HS Assay Kit (Invitrogen). Expression of DHRS9 and Ido1 mRNA was measured by RT-qPCR relative to GAPDH mRNA endogenous control using TaqPath 1-Step Multiplex Master Mix Kit (Applied Biosystems) and QuantStudio 5 Real-time PCR system (Applied Biosystems) according to manufacturer’s instruction. TaqMan assays were from Applied Biosystems: for Human Ido-1 assay number Hs00984148_m1 (FAM-MGB), for Human DHRS9 Hs00608375_m1 (FAM-MGB) and for Human GAPDH Hs03929097_g1 (VIC-MGB). Each amplification reaction contained 50 ng of RNA and was performed in triplicates. Data was analyzed with QuantStudio Design and Analysis desktop Software (Applied Biosystems). Changes in expression (Rq) were calculated relative to expression of corresponding mRNA in the starting material, i.e. CD14 + monocytes.

For Real-Time PCR experiments two million cells/culture-flasks of primary cortical cultures were harvested at the appropriate time-points and treatments. After washing twice with 5 ml ice cold 1×PBS, pH 7.4, cells were scraped in 1 ml of the same buffer and afterwards centrifuged for 5 min at 1000×g and 4°C. The resulting pellets were re-suspended in 0.6 ml OL1-buffer, containing 0.43 M ß-ME, from the Oligotex mRNA purification kit (Qiagen, Hilden, Germany) that was used for mRNA purification. For efficient cell disruption, the suspension was centrifuged for two minutes at maximum speed through QIAshredder homogenizers (Qiagen). The obtained mRNAs were immediately quantified and reverse transcribed into cDNA, using the Sensiscript RT kit (Qiagen) according to the manufacturers protocol. Reverse primer OdT18 (Promega) and random nanomers (Sigma, Taufkirchen, Germany) were used in separate reaction mixes. The success of the reverse transcription was verified via PCR reaction using specific primers for GAP-DH (not shown). Finally the corresponding cDNAs of one sample were combined and used as one template.

For quantitative real-time PCR, cell infection was conducted as described above. Isolation of total RNA from macrophages, NCI-H441 and homogenized lungs was done by using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Cells of the biochip model were first homogenized using the QIAshredder homogenizers (Qiagen) and total RNA was isolated with RNeasy Micro Kit (Qiagen) following manufacturer’s instructions. Equal amounts of RNA were transcribed into cDNA using QuantiNova Reverse Transcription Kit (Qiagen) according to the manufacturer’s protocol. Gene expression of several genes was determined by qRT-PCR on a Thermo Scientific™ PikoReal™ Real-Time PCR System using QuantiTect SYBR Green (Qiagen) and gene-specific primers (Table 1). Relative gene expression levels were referred to the housekeeping gene GAPDH for NCI-H441 and RPL37A for macrophages and calculated with the 2^−ΔΔCt method [30 (link)].
To analyze the mRNA expression of SP-A, we have compared the data from the infection with the pathogens and the data of the stimulation with recombinant TNF-α. For this, we used 10 nM of TNF-α (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) added to RPMI. After 30 min of incubation on NCI-H441 cells, they were washed with PBS and incubated for 4 h. Afterward, the mRNA expression was performed as described above.