Histopaque density gradient centrifugation

For in vitro experiments, heparinized venous blood samples from 10 of the 11 volunteers were collected and PBMCs were separated using Leucosep^TM-Tubes (Greiner Bio-One GmbH, Frickenhausen, Germany) and Histopaque^® density gradient centrifugation (Sigma Aldrich, Taufkirchen, Germany) [51 (link)]. In 6-well plates, 3.0 × 10⁶ cells per well were suspended in 2 mL RPMI-medium with 10% fetal calf serum, 1% L-Glutamin (Biochrom AG, Berlin, Germany), and 1% HEPES (Sigma Aldrich, Taufkirchen, Germany) for 24 h at 37 °C with 5% CO₂.

Streptavidin BV421 (405225; Biolegend, California, CA, USA) and biotinylated SARS-CoV-2 Spike RBD protein ((40592-V08H2-B; Sino Biological, Beijing, China) were incubated for 1 hour at 4°C to create the antigen probe. Peripheral blood mononuclear cells (PBMCs) were separated from heparinized blood base on the manufacturer’s recommendations using Histopaque density gradient centrifugation (10771, Sigma-Aldrich). After rinsing with FACS buffer (phosphate-buffered saline with 2% fetal bovine serum), staining was performed for 30 min at 4°C with an antigen probe (1:33.3) and the following binding antibodies: anti-human CD3 (300430, Biolegend), anti-human CD19 (302212, Biolegend), anti-human CD21 (354918, Biolegend), and anti-human CD27 (356406, Biolegend). These antibodies were added in 1:50 volume. After staining, cells were rinsed and resuspended in 150 µL FACS buffer. Subsequently, samples were evaluated using a flow cytometer (CytoFLEX, Beckman Coulter). FlowJo software version 10.0.7r2 was used for data analysis.

For PBMC isolation, 15 blood samples were collected into a K₃EDTA vacuum tube 2 weeks after the challenge. The PBMCs were prepared according to a previous study^{38 (link)}. Briefly, the collected blood samples were diluted with an equal volume of a balanced salt solution, and PBMCs were immediately isolated using Histopaque density gradient centrifugation according to the manufacturer’s instruction (Sigma–Aldrich). The diluted blood samples were mixed with half the volume of a Histopaque solution and then centrifuged at 400 × g for 35 min at room temperature. PBMCs were carefully aspirated from the Histopaque solution plasma interface.

Pancreatic islets were isolated from 8- to 12-week-old BALB/c mice (Jackson Laboratories, CA) and housed under conventional conditions in accordance with the Canadian Council on Animal Care. All experimental procedures were approved by the University of Alberta Research Ethics and Animal Use Committee (Study ID: AUP00000331). Prior to pancreatectomy, the common bile duct was cannulated and the pancreas was distended with 0.125 mg/mL cold Liberase TL Research Grade enzyme (Roche Diagnostics, Laval, QC, CA) in Hanks balanced salt solution (Sigma, St. Louis, MO, USA). Pancreas digestion was continued in a 37°C water bath for 14 minutes with light agitation. After the pancreatic digestion phase, islets were purified using histopaque-density gradient centrifugation (1.108, 1.083 and 1.069 g/mL, Sigma, St. Louis, MO, USA). Upon purification, islets were placed in Connaught Medical Research Laboratories (CMRL-1066) (Corning-cellgro, Manassas, VA, USA) supplemented with 10% fetal bovine serum, 1% L-glutamine (200 mmol/L, Sigma, St. Louis, MO, USA), 1% sodium pyruvate (100 mmol/L, Sigma, St. Louis, MO, USA), 1% non-essential amino acid 100x (Sigma, St. Louis, MO, USA) and 100 U/mL penicillin-G and100 μg/mL streptomycin (Sigma Aldrich Canada Co., Oakville, ON, CA) at 37°C/5%CO₂ at pH 7.4.

Adenocarcinoma SK-BR3 cell line (ATCC^® HTB-30™) and Raji cell line (ATCC® CCL-86™) were obtained from American Type Culture Collection. SK-BR3 was cultured in complete Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) and Raji was cultured in complete RPMI 1640 with 10% FBS. Human PBMCs were purified from anonymous healthy volunteers using histopaque density gradient centrifugation (Sigma-Aldrich). Human C1q (CompTech) and hFcRn:hβ2m (Novus Biologicals) were purchased. All primers were synthesized by Integrated DNA Technologies.