Leucosep

To generate erythroblasts, PD patients’ peripheral blood mononuclear cells (PBMC) were extracted from 10 ml of freshly extracted blood with the use of Lympholyte-H (Cedarline) and Leucosep polypropylene tubes (227290, Greiner) according to manufacturer’s indications. Briefly, blood was diluted in PBS at a 1:2 ratio and loaded on a 15 mL Lympholyte – Leucosep tube. Blood was centrifuged at 800 g for 25 min with no brakes at 4 C. Upon removal of the plasma, the PBMC enriched cell fraction was collected, washed several times with sterile PBS and upon PBMCs were cultured in StemSpan SFEM medium (Stemcell Technologies) containing 2 mM Ultraglutamine (Lonza), 1% Nonessential aminoacids (NEAA), 1% penicillin/streptomycin, 50 ng/ml Stem Cell Factor, 2 U/ml Erythropoietin, 1 uM Dexamethasone (Sigma), 10 ng/ml Interleukin-3 (R&D Systems), 10 ng/ml Interleukin-6 (R&D Systems), 40 ng/ml IGF-1 (R&D Systems) and 50 ug/ml Ascorbic Acid (Merck) for 6–9 days refreshing half of the medium every other day starting from day 2. Erythroblasts were isolated when reaching 60–70% of the total cell population by gradient centrifugation at 1000 × g for 20 minutes at room temperature over Percoll (GE Healthcare). Isolated erythroblasts were frozen in FBS containing 10% DMSO at −80 °C. Metabolic analysis was performed within 2 days after thawing.

Plasma samples were collected from 50 aged individuals who were born between 1928 and 1933 (85–89 years old) (Table 1). These individuals had participated in the Kawasaki Wellbeing Project, which recruits healthy individuals aged 85–89 years-old, from May to July 2018. Plasma samples from 33 younger adult volunteers born after 1962 (23–55 years old), who participated in a seasonal influenza vaccine surveillance project, were collected before these individuals received the seasonal influenza vaccine from January to November 2018 (Table 1). Information on the vaccination history of most of the individuals was limited. Each blood sample was collected in a 5-mL vacuum tube containing EDTA-2Na (Terumo, Tokyo, Japan). Plasma was separated from whole blood by using a Leucosep (Greiner Bio-One, Kremsmünster, Austria) and was stored at −20 °C until use.

Laboratory analyses were performed at Linköping University, Sweden. Blood and serum samples were collected at baseline and after 1, 2, 3, 6, and 15 months. Samples were drawn during the morning hours and peripheral blood mononuclear cells (PBMCs) were isolated within 24 h using Leucosep (Greiner Bio One) according to the manufacturer’s instructions.
Serum C-peptide was determined using a solid phase-two side enzyme immunoassay (Mercodia, Uppsala), and results were validated with the inclusion of a Diabetes Antigen Control Human (Low/High) (Mercodia, Uppsala, Sweden). Inter and intra assay variation were 7% and 4% respectively.

Laboratory analyses were performed at Linköping University, Sweden. Blood and serum samples from all the DIAGNODE-1 participants were collected at baseline and after 15, 30 months. Additionally, samples from the three patients in DIAGNODE Extension were collected at 31.5, 33.5, 36.5 and 42.5 months. Samples were drawn during the morning hours, and peripheral blood mononuclear cells (PBMCs) were isolated within 24 h using Leucosep (Greiner Bio One) according to the manufacturer’s instructions.
Analysis of serum C-peptide was performed using a solid-phase two-sided enzyme immunoassay (Mercodia, Uppsala). Results for each assay were validated with the inclusion of a Diabetes Antigen Control Human (Low/High) (Mercodia, Uppsala, Sweden). The assay is calibrated against the international reference reagent for C-peptide IRR C-peptide 84/510. Inter- and intra-assay variations were 6.6% and 3.5%, respectively.

For serum, fresh blood was collected into a preservative-free vacutainer tube and allowed to clot for at least 30 min at room temperature before being centrifuged for 20 min at 1300 x g at room temperature. The serum was then stored at −80°C until analysis. For peripheral blood mononuclear cells (PBMCs), fresh blood was collected into heparinized vacutainer tubes and peripheral mononuclear cells were separated using LeucoSep (Greiner Bio-One) tubes. Cells were washed twice with phosphate buffered saline (PBS), counted and resuspended in freezing media containing 90% fetal bovine serum (FBS) and 10% DMSO at 5–20×10⁶/mL. Cells were frozen in liquid nitrogen until further analysis.

Peripheral Blood Mononuclear Cells (PBMCs) were immediately isolated by density centrifugation using pre-filled Greiner Bio-One Leucosep® tubes according to the manufacturer’s recommendations. PBMCs rings were collected and washed twice in PBS before final suspension at 20 × 10⁶ cells/ml in ice-cold freezing medium (50% RPMI-1640, 40% fetal bovine serum, 10% DMSO) and transferred at −80°C in Nalgene® Mr. Frosty for short-term storage (<1 month). For monocyte isolation, 40 × 10⁶ to 60 × 10⁶ cells of cryopreserved PBMCs were thawed and quickly washed with 13 ml ice-cold RPMI-1640. Median cell viability upon recovery was assessed by trypan blue exclusion (88.37%, IQR: 82.1-97). Pan- and CD16+ monocyte purification were performed using Pan Monocyte Isolation Kit and CD16+ Monocyte Isolation Kit respectively according to manufacturer’s instructions (Miltenyi Biotec GmbH).