96 well flat bottom plate

Huh7.5 cells were plated at 8000 cells per well of flat bottom 96-well plates (Thermo Fisher Scientific, Roskilde, Denmark). The next day, cells were inoculated with SARS-CoV-2/human/Denmark/DK-AHH1/2020 at MOI 0.0198 by adding 50 µL of diluted virus stock per well. MOI were chosen to yield obviously infected cultures with counts of infected cells in the target range given in the section on immunostaining and evaluation of 96-well plates below. In addition, MOI were selected to avoid virus induced cytopathic effects during the assay. Directly after viral inoculation, 50 µL of medium containing extracts or compounds were added resulting in the specified concentrations; alternatively, 50 µL of medium containing diluent (DMSO) were added resulting in the specified dilutions. In each independent experiment, each concentration was tested in seven replicates; 14 infected and nontreated as well as 12 noninfected and nontreated control wells were included in the assay. After 72 ± 2 h incubation at 37 °C and 5% CO₂, cultures were immunostained for SARS-CoV-2 spike glycoprotein and evaluated as described below.

Serum was collected from each mouse one week post each immunization. The EmCRT-specific IgG and subtype IgG1 and IgG2a were measured in these sera using an indirect enzyme-linked immunosorbent assay (ELISA). Briefly, flat-bottom 96-well plates (Thermo Fisher, Waltham, MA, USA) were coated with 100 μL/well of rEmCRT at a concentration of 1.0 μg/mL in bicarbonate buffer (pH 9.6) overnight at 4 °C. After three washes with PBS + 0.05% Tween-20 (PBST), the plates were blocked with blocking buffer (5% BSA in PBS) for 1 h at 37 °C, then probed with serial dilutions of mouse sera for 1 h at 37 °C. After being washed 3 times with PBST, the plates were incubated with HRP-conjugated goat anti-mouse IgG or IgG1 or IgG2a (Invitrogen, Carlsbad, CA, USA) for 1 h at 37 °C. After the final wash, the substrate 3, 3’, 5, 5’-tetramethylbenzidine (TMB, Beyotime Biotechnology, Shanghai,, China) was added to each well, and the reaction was stopped with stop solution (Beyotime Biotechnology, Shanghai,, China). Quantification of the reaction was determined by measuring the absorbance at 450 nm in an ELISA reader [30 (link),31 (link)].

24h prior to differentiation, mESCs were induced by passaging into IMDM based media supplemented with LIF. To start the differentiation culture (d0), cells growing in IMDM were trypsinized and plated in differentiation media in either triple vent petri dishes (Thermo Fisher) or flat-bottom 96-well plates (Thermo Fisher) at 3x10⁴ cells in 10 cm dishes or 100–1200 cells per well of a 96-well plate for up to seven days without further intervention except for daily gentle shaking of the dishes to disrupt potential EB attachment to the bottom of the dish. EBs were harvested and disaggregated in 0.25% trypsin for 3 minutes at 37°C. See S1 Methods for details.
CD71+(high) cells were isolated by magnetic column separation (LS Column, Miltenyi), according to the manufacturer’s instructions. Briefly, cells from disaggregated EBs were labelled with anti-mouse CD71-FITC (eBioscience 11-0711-85; 1:200) in staining buffer (PBS with 10% FCS; 500 μl per 10⁷ cells) for 20 minutes at 4°C, washed, then incubated with MACS anti-FITC separation microbeads (Miltenyi; 10 μl per 10⁷ cells, according to manufacturer’s instructions). Bead-labelled cells were retained by LS Columns. See S1 Methods for details.

Frozen isolates were used to inoculate into 5 mL of tryptic soy broth (TSB; Bectin Dickinson and Company, Franklin Lakes, NJ, USA). The conical tube containing the inoculate was incubated overnight at 37 °C with shaking at 250 rpm. After 16 h of incubation, Mueller Hinton broth (MHB; Bectin Dickinson and Company) was used to dilute all strains to 0.5 × 10⁶ CFU/mL using the 0.5 MacFarland Standard (GFS Chemicals, Columbus, OH, USA) in an Infinite M200 Spectrophotometer (Tecan, Männedorf, Switzerland). High throughput methods were used for experiments in sterile, tissue culture treated, flat bottom 96-well plates (Thermo Fisher Scientific, Waltham, MA, USA). All trials were performed in triplicate using fresh, independent inoculates for each culture. Two lab strains of S. epidermidis ATTC 12228 [27 (link)] and ATTC 35894 [28 (link)], as well as nine clinical isolates from PJI in arthroplasty infections were tested. Clinical isolates from PJI patients were obtained from a clinical testing laboratory from cultures prepared on TSB agar slants. S. epidermidis clinical isolates were then grown in TSB overnight with shaking and stored in cryotubes at −80 °C in TSB with 10% glycerol to create an isolate library bank. All procedures for this study were followed according to Institutional Review Board (IRB) guidelines and regulations, IRB approval #PRO15070263.

A549-hACE2 cells were plated at 10,000 cells per well of flat bottom 96-well plates (Thermo Fisher Scientific, Roskilde, Denmark). The next day, cells were inoculated with SARS-CoV-2/human/Denmark/DK-AHH1/2020 at MOI 0.005 by adding 50 µL of diluted virus stock per well. The virus containing supernatants were removed after 2 h incubation, and wells were washed once with 130 µL PBS. Directly after, 50 µL of medium were added. At different timepoints during the experiment, 50 µL of medium containing artesunate were added resulting in a final concentration of 14 µg/mL: 0 h post inoculation, addition at the time of viral inoculation with presence of the drug during the 2 h viral infection phase; 2 h, addition 2 h post inoculation, immediately after the 2 h viral infection phase; 4 h and 6 h, addition 4 h and 6 h post inoculation, respectively. During the experiment, cultures were incubated at 37 °C and 5% CO₂. 48 ± 2 h after inoculation, cultures were immunostained for SARS-CoV-2 spike glycoprotein and evaluated as described below. 96-well images from this experiment in A549-hACE2 cells are shown in Figure S11.

Naive (CD4⁺CD62L⁺CD44⁻CD25⁻) T-cell were isolated by cell-sorting and stimulated with 50 ng/well of plate-bound anti-CD3 and 2 μg/mL soluble anti-CD28 Abs on flat-bottom 96-well plates (Thermo Scientific) for 5 days. To force the differentiation of the different Th phenotypes, naïve CD4⁺ T-cells were incubated in the conditions indicated above, in addition to a mixture of cytokines and blocking antibodies: Th1: 20 ng/mL IL-12, 10 ng/mL IL-2 and 5 μg/mL anti-IL-4; non-pathogenic Th17 (Th17np): during the first 2 days with 25 ng/mL IL-6, 5 ng/mL TGF-β1, 20 ng/mL IL-1β, 5 μg/mL anti-IL-4, 5 μg/mL anti-IL-2 and 5 μg/mL anti-IFN-γ and during the last 3 days with 25 ng/mL IL-6, 5 ng/mL TGF-β1; pathogenic Th17 (Th17p): during the first 2 days with 25 ng/mL IL-6, 5 ng/mL TGF-β3, 20 ng/mL IL-1β, 5 μg/mL anti-IL-4, 5 μg/mL anti-IL-2 and 5 μg/mL anti-IFN-γ and during the last 3 days with 25 ng/mL IL-6, 20 ng/mL IL-1β and 20 ng/mL IL-23. At different incubation times, cells were assessed for gene expression real time RT-PCR.