Neutral red solution

VZV plaque reduction assays were performed as described previously^{31 (link)}. Briefly, human foreskin fibroblast (HFF) cells were seeded into 6-well plates. After seeding (48 h), they were infected with virus at a concentration that would yield 25–30 plaques per well. After 1 h incubation, the drug was serially diluted 1:5 at 6 different concentrations and added to the wells. The plates were incubated for 10 days, stained with a 1% neutral red solution (Sigma), and counted using a stereomicroscope. 50% effective concentration (EC₅₀) and 50% cytotoxic concentration (CC₅₀) values were calculated using the MacSynergy program.

A plaque reduction VN assay was performed as described previously (83 (link)) with modifications. Serum and whey were inactivated at 56°C for 30 min prior to testing for PEDV neutralizing Abs. Serial 4-fold dilutions of test sera or whey were mixed with 70 PFU and 140 PFU PEDV PC22A cell-culture adapted strain, respectively. The serum/whey-virus mixtures (290 μl/well) were incubated for 1.5 h at 37°C with gentle rocking and then duplicate samples were infected onto 2–3 day confluent Vero cell monolayers in 6-well plates. Plates were incubated at 56°C for 1 h, lightly rocking every 15 min. Subsequently, the inoculum was removed, cells were washed twice with sterile PBS [1X pH 7.4 (Sigma Aldrich, St. Louis, MO)] and overlaid with 0.75% low melting point agarose (SeaPlaque, Lonza, Riverside, PA) in serum free media supplemented with tryptose phosphate broth and trypsin as described for PEDV cultivation (75 (link)). Plates were incubated at 37°C for 3 days and then stained with 0.001% neutral red solution (Sigma Aldrich, St. Louis, MO). Plaques were counted and the VN titers were determined by calculating the reciprocal of the highest dilution of a serum/whey sample showing an 80% reduction in the number of plaques compared with seronegative control serum/whey.

Vero cells were plated at 6 × 10⁵ cells/well in 6-well plates and incubated overnight. The plasma samples were incubated at 56 °C for 30 min to inactivate the complements, serially diluted from 10¹ to 10⁷ folds with pre-warm, serum-free DMEM. The diluted samples were mixed with 100 PFUs of ZIKV (strain PRVABC59) and incubated at 37 °C for 1 h. The virus-plasma mixtures were applied onto Vero cells and incubated for 1 h. Then the virus-containing medium was replaced with DMEM overlay medium containing 1% SeaPlaque Agarose (Lonza), 10% FBS, and 1% P/S and incubated for 4 days. The plaques were stained with 0.1% Neutral red solution (Sigma) and counted^{68 (link)}. Then the PRNT₅₀ values were calculated with GraphPad Prism software (version 7.0)^{72 (link)}.

VeroE6/TMPRSS2 cells (1.8 × 10⁵) were seeded in a 24-well plate with culture medium (DMEM high glucose medium supplemented with 10% FCS, 1% L-Glutamine, 1% Penicillin/Streptomycin; all reagents were obtained from Sigma Aldrich, Missouri, USA) and incubated overnight at 37°C and 5% CO₂. On the following day, whole serum or plasma samples were serial-diluted from 1:1 to 1:6,250 and incubated with SARS-CoV-2 strains (1.5 × 10⁴ PFU/ml) for 1 h at 37°C. After incubation serum/plasma-virus mix was ultracentrifuged and resuspended in DMEM high glucose medium supplemented with 5% FCS, 1% L-Glutamine, 1% Penicillin/Streptomycin. VeroE6/TMPRSS2 cells were then inoculated with antibody-opsonized SARS-CoV-2 for 30 min on a shaker at RT and 30 min in an incubator at 37°C and 5% CO₂. After incubation, inoculation medium was replaced with culture medium containing 1.5% Low-melt Agarose (Biozym, Oldendorf, Germany). Cells were incubated for 3 days at 37°C and 5% CO₂ before plaque visualization and counting using 0.1% Neutral Red solution for 3 h (Sigma Aldrich, Missouri, USA).

The infectivity of MNV-1 was determined by a plaque assay, as described previously [9 (link)]. Briefly, RAW264.7 cells were seeded into 6-well plates (Falcon BD, Franklin Lakes, NJ) at approximately 6 log cells/mL in DMEM containing 5% (v/v) FBS, penicillin (100 U/mL), and streptomycin (100 μg/mL). The plates were incubated at 37°C under 5% CO₂ for 18 h. Then, 500 μL/well of samples prepared as described below sections were added to the plates, and the plates were shaken at 20 r/min for 1 h. The inoculated sample was removed, and the wells overlaid with 2 mL of 1.5% (v/v) SeaPlaque agarose (Lonza Japan, Tokyo, Japan) in DMEM containing 5% (v/v) FBS. The plates were incubated at 37°C under 5% CO₂ for 48 h. Then, 2 mL of 0.03% (v/v) neutral red solution (Sigma-Aldrich Japan, Tokyo, Japan) was added, and the plates incubated at 37°C under 5% CO₂ for 1 h. Thus, visualized plaques were then counted.

All SARS-CoV-2 experiments were performed in a laboratory of biosafety level 3.
SARS-CoV-2 isolates from respiratory specimens of four different patients: Alpha variants SARS-CoV-2/hu/Germany/Jena-vi005159/2020 [isolate 5159] (MW633322.1), SARS-CoV-2/hu/Germany/Jena-vi005587/2020 [isolate 5587] (MW633323.1) and SARS-CoV-2/hu/Germany/Jenavi005588/2020 [isolate 5588] (MW633324.1) [44 (link)] and Delta variant SARS-CoV-2/hu/Germany/Jena-0114749/2021 [isolate 4749] (ON650061) were used for infection experiments. The isolate 5159 belongs to the B.1-lineage, the isolates 5587 and 5588 to the B.55-lineage and the isolate 4749 to the AY.126-lineage.
For plaque assay, Vero-76 cells were seeded in 6-well plates and infected with serial dilutions of the supernatants in PBS supplemented with 1 mM MgCl₂, 0.9 mM CaCl₂, 0.2% BSA and 100 U mL⁻¹ Pen/Strep (Sigma-Aldrich, Taufkirchen, Germany) for 90 min at 37 °C. After aspiration, the cells were incubated with 2 mL MEM supplemented with 0.9% agar (Oxoid, Wesel, Germany), 0.01% DEAE-Dextran (Pharmacia Biotech, Freiburg im Breisgau, Germany), 0.2% BSA and 0.2% NaHCO₃ (Sigma-Aldrich, Taufkirchen, Germany) at 37 °C and 5% CO₂ for 3 days. To visualize the plaques, a staining with neutral red solution (Sigma-Aldrich, Taufkirchen, Germany) in PBS was performed and the number of infectious particles (pfu mL⁻¹) was determined by counting.

Neutral red staining was used to observe the macrophage, following published procedures (Herbomel et al., 2001 (link); Davis and Ramakrishnan, 2009 (link); Shiau et al., 2015 (link)). The 3 dpf zebrafish were wounded consistently by tailfin transection with a sterile scalpel. Subsequently, zebrafish were immersed in neutral red solution (2.5 μg/mL + 0.003% PTU) (Sigma Aldrich) at 28°C in the dark for 7–9 h. Furthermore, in order to observe the response of macrophages against mycobacterial infection, Mm were injected into the hindbrain of 30–32 hpf zebrafish embryos with 50 CFU according to the previous method (Gutzman and Sive, 2009 (link)). Zebrafish were then incubated in neutral red solution as already described. Anesthetized zebrafish were embedded into solidified agarose drops and viewed under light microscopy to acquire images (SteREO Discovery.V20; Carl Zeiss, Oberkochen, Germany).