Ultra 4 centrifugal filter unit

scFv-Clasp (~1 mg/ml) was adjusted to pH 8.3 with 1 M NaHCO₃, incubated either with Alexa Fluor (AF) 647 NHS ester (Invitrogen, Cat.# A20106) or CF568 NHS ester (Sigma-Aldrich, Cat.# SCJ4600027), according to manufacturer’s instructions, for 1.5–2 h while shaking at room temperature in the dark. Subsequently, the solution was centrifuged for 10 min at 10,000 g at 4 °C. If necessary, the volume was adjusted to 500 µl with PBS, and loaded on an equilibrated G-25 column (Column PD Minitrap G-25, GE Healthcare, Cat.# 28-9180-07). Once the solution fully entered the column, conjugated scFv-Clasp was eluted with 1 ml PBS. To remove any residual unbound dye, the eluate was washed 3 times with PBS in an Amicon Ultra-4 Centrifugal Filter Unit (NMWL 3 kDa, Millipore, UFC800324) and finally concentrated to <250 µl. The degree of labelling, assessed photometrically, was 5.5 for AF647-scFv-Clasp and 2 for CF568-scFv-Clasp.

C. jejuni OMVs were isolated as previously described (Elmi et al., 2012 (link)). Briefly, C. jejuni from a 24 h BA plate were resuspended in Brucella broth and used to inoculate 50 ml of pre-equilibrated Brucella broth to an OD₆₀₀ of 0.1. Cultures were grown to late-log phase, time points determined by growth kinetics for each strain. OD₆₀₀ values were normalized to OD₆₀₀ 1.0 and the sterile supernatants obtained by pelleting cells and filter-sterilizing supernatants through a 0.22 μm membrane (Millipore, United Kingdom). The supernatants were then concentrated to 2 ml using an Ultra-4 Centrifugal Filter Unit with a nominal 10 kDa cut-off (Millipore). The concentrated filtrate was ultra-centrifuged at 150,000 × g for 3 h at 4°C using a TLA 100.4 rotor (Beckman Instruments, United States). The resulting OMV pellet was washed by resuspending in phosphate buffered saline (PBS) and pelleting by ultra-centrifugation as described above. All isolation steps were performed at 4°C and the resulting OMVs pellet was resuspended in PBS and stored at −20°C.

MIN-6 (1 × 10⁷), INS-1 (1 × 10⁷), and bEND.3 (5 × 10⁶) cells were briefly dispersed with complete media and attached for 24 hrs in a 100 mm cell culture dish. To expose cells to stress conditions, the culture media was exchanged with DMEM (Hyclone # SH30021) containing low glucose (1,000 mg/L) supplemented with 1% FBS and antibiotics and maintained for an additional 72 hrs in the same incubator. Culture supernatants were collected, centrifuged at 1,200 rpm for 5 min to eliminate the pellet, passed through a 0.45 μm syringe filter to obtain standard CM, and stored in aliquots at −80 °C for future use. CM from islets was prepared by same procedures from culture supernatant of isolated rat islets maintained in 2,000 IEQ/ml for 72 hrs in stress-inducing media, as previously mentioned. In some experiment MP-deprived CM was used instead. MP- depleted CM was prepared by removing large particles from the standard CM using centrifugal filter units (Amicon Ultra-4 Centrifugal Filter Unit with an Ultracel-100 membrane, Millipore #UFC810008).

Proteins and their peptides were extracted from both the EAT and EAT secretome of 3 patients with and 3 without AF by liquid chromatography-tandem mass spectrometry using an Ultimate 3000 Nano LC–MS/MS system (Dionex LC-Packings, Amsterdam, The Netherlands) and identified as described previously [20 (link), 29 (link)]. Specific to this study, secretome extracted from ± 20 1 mm EAT cubes per sample were concentrated approximately 40 times using ultrafiltration devices (Amicon Ultra-4 Centrifugal Filter Unit, millipore, UFC801024), and EAT samples were cryo-milled and solubilized before protein denaturation and fixation (gels: Additional file 1: Fig. S1). For protein identification, MS/MS spectra were searched against the Swiss-Prot human FASTA file (canonical and isoforms, downloaded March 2017, 42161 entries) using MaxQuant version 1.5.4.1.[9 (link)]. Proteomes from EAT and EAT secretome were uploaded to the ProteomeXchange Consortium via the proteomics identification database PRIDE with accession number PXD013230.

Pleural effusion samples were depleted of six high-abundance human plasma proteins (albumin, immunoglobulin G, immunoglobulin A, transferrin, α1-antitrypsin, and haptoglobin) using a Multiple Affinity Removal System (MARS) affinity column (Hu-6HC, 4.6 × 100 mm; Agilent Technologies, Wilmington, DE, USA) on an ÄKTA Purifier-10 fast performance liquid chromatography system (FPLC; GE Healthcare/Amersham Bioscience, UK). Depleted pleural effusion samples were concentrated using an Amicon Ultra-4 centrifugal filter unit with an Ultracel-3 membrane (Millipore, Carrigtwohill, Co. Cork, Ireland). After the depletion of high-abundance proteins, the protein concentrations of the PPE samples were determined using the BCA protein assay kit from Pierce (Rockford, IL, USA). In the experiment 1, pleural effusion samples (10 μg proteins) from each 4 UPPE patients and 4 CPPE patients with high-abundance protein depletion were then pooled into UPPE group (total 40 μg proteins in UPPE) and CPPE group (total 40 μg proteins in CPPE), respectively. For the technical replicates, the second set (experiment 2) of pooled samples from 4 UPPE patients and 4 CPPE patients were prepared from different batches.

Viral nucleic acid was isolated using the Quick DNA/RNA Viral Kit (Zymo Research, catalog# D7020) according to the manufacturer’s instructions. For buffy coat cells, approximately 2 × 10⁶ cells were washed briefly with 1.5 mL Hanks Balanced Salt (HBSS, Corning #21-022-CV) solution to remove dimethyl sulfoxide (DMSO). Washed cells were pelleted by centrifugation (400× g, 10 min), the supernatant removed, and the pellet resuspended in 200 μL HBSS. Viral nucleic acid was extracted with the High Pure Viral Nucleic Acid Kit (Roche, Basel, Switzerland, catalog #11858874001) according to the manufacturer’s instructions. For co-culture experiments, the media was concentrated 10-fold (2 mL to 200 µL) with an Amicon Ultra-4 centrifugal filter unit (Millipore-Sigma #UFC80500) as per the manufacturer’s instructions. Nucleic acid was isolated from the concentrated sample with the High Pure Viral Nucleic Acid Kit (Roche) according to the manufacturer’s instructions. All nucleic acid samples were stored at −80 °C.