Jurkat lucia nfat cd16 cells

The ability of plasma antibodies to cross-link spike SARS-CoV-2 protein and activate FcγRIIIa on Jurkat-Lucia™ NFAT-CD16 cells (Invivogen, USA) was measured as a proxy for ADCC. High-binding 96-well plates were coated with 1 μg/ml spike SARS-CoV-2 protein and incubated at 4°C overnight. Plates were then washed with PBS and blocked at room temperature for 1h with 2.5% bovine serum albumin (BSA)/PBS. After washing, heat-inactivated plasma (1:100 final dilution) or monoclonal antibodies (final concentration of 100 μg/ml) in RPMI media supplemented with 10% FBS 1% Pen/Strep were added to the wells and incubated for 1h at 37°C. Jurkat-Lucia™ NFAT- CD16 2 × 10⁵ cells per well were added and incubated for 24h at 37°C and 5% CO₂. Twenty microliter of supernatant was then transferred to a white 96-well plate with 50 μl of reconstituted QUANTI-Luc secreted luciferase and read immediately on a Victor 3 luminometer with 1 s integration time. The RLUs of a no antibody control were subtracted as background. Palivizumab was used as a negative control, while CR3022 was used as a positive control, and P2B-2F6 to differentiate the Beta from the D614G variant. To induce the transgene, 1× cell stimulation cocktail (Thermo Fisher Scientific, Oslo, Norway) and 2 μg/ml ionomycin in R10 was added. RLUs for spikes were normalized to each other and between runs using CR3022.

SARS-CoV-2 spike-expressing CEM cells were generated by transfection with linearized plasmid (pcDNA3.1) encoding codon-optimized full-length SARS-CoV-2 spike protein matching the amino acid sequence of the IL-CDC-IL1/2020 isolate (GenBank, ACC# MN988713) and B.1.1.529. Spike-expressing CEM cells were plated at 100,000 per well in round bottom 96-well plates and incubated with 100 μl of diluted plasma (100-fold) for 30 min at 37°C. Cells were washed, and 200,000 Jurkat-Lucia NFAT-CD16 cells (Invivogen) were added to each well in 100 μl of Iscove’s Modified Dulbecco’s Medium (IMDM) with 10% FBS. The cells were then centrifuged for 1 min at low speed and co-cultured for 24 hours at 37°C. Quanti-Luc (50 μl) was added to 20 μl of coculture supernatant, and luminescence was measured immediately on a luminometer (2104 Multilabel reader, PerkinElmer).

HEK293F suspension cells were cultured in 293 Freestyle media (Gibco BRL Life Technologies, Ontario, CA) and incubated shaking at 37°C, 5% CO₂ and 70% humidity at 125 rpm. HEK293T cells were cultured at 37°C, 5% CO₂, in DMEM (Gibco BRL Life Technologies, Ontario, CA) containing 10% heat-inactivated fetal bovine serum (FBS) and supplemented with 50 μg/ml Gentamicin. Cells were disrupted at confluence with 0.25% trypsin in 1 mM EDTA every 48h–72h. HEK293T/ACE2.MF cells were maintained as for HEK293T cells but were supplemented with 3 μg/ml Puromycin for selection of stably transduced cells. THP-1 cells were used for the ADCP assay and obtained from the AIDS Reagent Program, Division of AIDS, NIAID, NIH contributed by Dr. Li Wu and Vineet N. Kewal Ramani. Cells were cultured at 37°C, 5% CO₂ in RPMI (Gibco BRL Life Technologies, Ontario, CA) containing 10% heat-inactivated FBS with 1% Penicillin-Streptomycin (Pen/Strep) and 2-mercaptoethanol to a final concentration of 0.05 mM and not allowed to exceed 4 × 10⁵ cells/ml to prevent differentiation. Jurkat-Lucia™ NFAT-CD16 cells (Invivogen, USA) were maintained in IMDM (Gibco BRL Life Technologies, Ontario, CA) media with 10% heat-inactivated FBS, 1% Pen/Strep, and 10 μg/ml of Blasticidin and 100 μg/ml of Zeocin were added to the growth medium every second passage to allow the selection of CD16 expressing cells.