Molecular grade ethanol

Extraction was performed using a
PureLink Viral RNA/DNA Mini Kit (Invitrogen) according to manufacturer’s
instruction. Briefly, 25 μL of Proteinase K was added, before
adding 200 μL of Viral Lysis Buffer. Samples were briefly vortexed
and incubated at 56 °C for 15 min. 250 μL of molecular
grade ethanol (Merck Life Sciences) was then added to each sample
before a brief vortexing and incubation for 5 min at room temperature.
Samples were then added to a Viral Spin Column in a collection tube
before being centrifuged in a benchtop microcentrifuge at 6800g for 1 min (all subsequent centrifugation steps assume
this speed unless otherwise specified). The flow-through was discarded,
and 500 μL of wash buffer (WII) was added, before being centrifuged
for 1 min. The flow-through was discarded again, and another 500 μL
of wash buffer was added to each sample, before being centrifuged
for 1 min. The spin column was then centrifuged in a clean collection
tube for 1 min to remove any residual wash buffer (WII). Each spin
column was then placed in a collection tube, and genetic material
was eluted with 50 μL of sterile Rnase-free water. Samples were
then incubated at room temperature for 1 min before being centrifuged
for 1 min to elute nucleic acids. Purified viral DNA was stored at
−80 °C before being amplified in qPCR.

Total RNA was extracted from each tissue block embedded in paraffin up to two blocks per organ to perform the viral amplicon amplification and sequencing. For RNA extraction, 10 µm thick sections were performed on the tissue block until 100 µm per block was obtained. Subsequently, the sections were placed in a 1.5 mL tube with 1 mL of xylol (Merck, USA), centrifuged at 15,000 rpm for 1 min, vigorously stirred, and centrifugation was repeated for 1 min. Subsequently, 1 mL of molecular grade ethanol (Merck, USA) was added and centrifuged at 15,000 rpm for 1 min. Then, the ethanol was carefully removed and allowed to evaporate. Next, 500 µL of lysis buffer with proteinase K (10 mM Tris–HCl, 2 mM EDTA, 1% SDS; 0.2 µg/µL proteinase K) was added and incubated at 56 °C for 24 h [28 (link)]. Subsequently, the mixture was incubated at 80 °C for 15 min and left at − 20 °C for 3 min. From this step, RNA extraction was continued using the RNeasy Mini-kit (Qiagen,Germany) following the protocol recommended by the manufacturer. The RNA obtained was stored at − 80 °C for later use.

For the enzyme-linked immunospot (ELISpot) assay, multiScreen-IP filter plates (Merck Millipore, Burlington, MA, USA) were pre-wetted with 70% molecular grade ethanol (Sigma-Aldrich), washed and coated with PBS, 0.5 μg/ml goat anti-human IgG (Newmarket Scientific, Newmarket, UK), 0.8 or 1.6 μg/ml GAD (RSR, Dallas, TX, USA), 0.8 or 1.6 μg/ml islet antigen-2 (IA-2) (RSR), or 10 or 50 μg/ml proinsulin (Biomm, Nova Lima, Brazil) overnight at 4°C. Plates were washed and blocked with 10% heat-inactivated FBS/RPMI overnight at 4°C. Stimulated cells were washed in 10% heat-inactivated FBS/RPMI, resuspended at 5 × 10⁵ cells/ml (for the IgG positive control) or 4 × 10⁶ cells/ml (for all other conditions), and plated at 100 μl/well (n ≥ 6 wells per condition). Plates were incubated for 5 h at 37°C, washed in 0.05% Tween 20/PBS and coated with 0.5 μg/ml goat anti-human IgG Fc biotin in 5% heat-inactivated FBS/PBS overnight at 4°C. Antibody secretion was revealed using ExtrAvidin (Sigma-Aldrich) followed by BCIP/NBT substrate (Sigma-Aldrich). Plates were washed and dried overnight and spots were counted using a BIO-SYS Bioreader 4000 (BioSys, Miami, FL, USA). The PBS background was subtracted from all counts (mean±SD background 142 ± 75, range 30.5–245, not significantly different between healthy donors and slow progressors).

The active metabolite of the SSRI escitalopram, (S)‐citalopram, has a therapeutic window of between 50 and 130 ng/ml (Kumar, Kung, & Shinozaki, 2014), which corresponds to doses of between 120 and 313 nM in vitro. Subsequently, cells were treated with a range of doses, incorporating this therapeutically‐relevant window. Drug doses were achieved by dissolving escitalopram oxalate (Sigma) in molecular grade ethanol (Sigma) to form a 10 mM stock solution. Drug doses (145 nM, 290 nM, and 1160 nM) were then formed by dilution of the stock with media; with the relative proportion of ethanol kept constant across all dose groups including a vehicle control (0 nM).

After five days of growth on etched coverslips in duplicate wells, AR42J-B13 cells were rinsed with PBS and immersed in an incremental gradient of 30, 50, 70, 90 and 100% molecular grade ethanol (Sigma), diluted in PBS. Samples were then coated with platinum and visualised with the assistance of Elaine Miller at Curtin University’s Microscopy and Microanalysis Facility on a Zeiss Neon 40EsB FIBSEM (Zeiss Australia, North Ryde, NSW, Australia).

25-hydroxyvitamin D3-deuterated 3 used for internal standards was ordered from Cambridge Isotope Laboratories (Tewksbury, MA, USA). 1,25-dihydroxyvitamin D3-d3 was used as another internal standard (Sigma Aldrich). 24R,25-dihydroxyvitamin D3-d6, 25-hydroxyvitamin D3 monohydrate-d6, and 1,25-dihydroxyvitamin D3-d6 were used for the calibration standard curves (Sigma Aldrich). Liquid chromatography–mass spectroscopy (LCMS)-grade methanol and water were used to make mobile phases and to extract and purify samples (Thermo Fisher Scientific, Waltham, WA). Ammonium acetate, formic acid, and molecular grade ethanol were used to make mobile phases (Sigma Aldrich).