Advanced rpmi 1640

Jurkat human T lymphocytic leukemia cell line was purchased from the American Type Culture Collection, (ATCC, Manassas, VA, USA). Jurkat cells were cultivated in suspension in Advanced RPMI 1640 (Gibco/Invitrogen, Paisley, UK) supplemented with 10% fetal bovine serum (FBS) (Gibco/Invitrogen, Paisley, UK) and 10 mM Hepes, 1 mM of sodium pyruvate, and 1% Pen/Strep 5000 U/mL (Gibco/Invitrogen, Paisley, UK).
THP-1 human monocytic leukemia cell line (ATCC, Manassas, VA, USA) was cultivated in Advanced RPMI 1640 (Gibco/Invitrogen, Paisley, UK) supplemented with 10% fetal bovine serum (Gibco/Invitrogen, Paisley, UK) and 1% Pen/Strep 5000 U/mL (Gibco/Invitrogen, Paisley, UK). Cell cultures were maintained at 37 °C with 5% CO₂ and examined daily under a light microscope for quality control.

For the secretome experiments, pDCs were enriched from leukopaks obtained from the New York Blood Center. First, PBMCs were extracted over Ficoll, and then, pDCs were enriched using EasySep Human Plasmacytoid DC Enrichment Kit [Cat # 19062]. pDCs were cultured in a 1:1 mixture of Advanced RPMI-1640 [Gibco 12633012] and RPMI-1640 [Gibco 11875119], without serum supplementation in ultra-low protein binding 24-well plates. Where indicated, influenza virus H1N1 A/PR/8/34 [Advanced Biotechnologies] at 0.4 HAU/mL was used for stimulation.
For pDC scRNA-seq, human donors were recruited from the Genotype and Phenotype Registry [GaP], a living biobank of genotyped individuals who have volunteered for recall on the basis of their genotype (33 (link)). GaP donors are genotyped on the Illumina Global Screening Array (GSA). Demographic data on the GaP donors can be found in Table S4. 100CC fresh whole blood was drawn from these donors into sodium-heparin vacutainers. PBMCs were extracted over Ficoll. pDCs were isolated from PBMCs using Miltenyi Plasmacytoid Dendritic Cell Isolation Kit II, human [Cat# 130-092-207]. pDC purity was determined by flow cytometry against BDCA2 and/or BDCA4. pDCs were cultured in Advanced RPMI-1640 [Gibco 12633012] supplemented with 5% FCS, with the same concentration of influenza virus as in the secretome experiment.

Single splenocyte suspensions were prepared as described above. The medium was changed to advanced RPMI 1640 (Gibco, 12633020). AO/PI dye (CountStar, RE010212) was used to determine the density and viability of splenocyte. The splenocytes and Sp2/0 cells were mixed at a ratio of 5:1. Electrofusion solution (BTX, 47-0001) was used to resuspend the mixed cells for cell fusion. The cell fusion instrument (BTX, ECM2001) was set to a voltage of 850 V and pulse of 40s. The fused cells were cultured in advanced RPMI 1640 medium (containing 10% FBS, 1% P/S) for 10 h (37°C, 5% CO₂) for regeneration. Semi-solid medium (StemCell, 03804) was used for the culture of hybridoma cells for 10 days. Microscopically opaque cell colonies were picked using a 10 μl pipette tip and cultured in advanced RPMI 1640 medium containing HT supplement (Gibco, 11067-030). After 3–5 days, the supernatant of hybridoma cells with a confluence of 50–70% was collected for ELISA screening. GP1 at 0.5 mg/L was used for ELISA coating. The supernatant of hybridoma cells was used as the test sample. The secondary antibody (1:5000) was an HRP-conjugated mouse anti-human IgG Fab antibody (GenScript, A01855). The colour reaction was conducted at room temperature and terminated after 20 min.