Crystal violet

The neutralization capacity of serum samples against a SARS-CoV-2 clinical isolate from September 2020 with the D614G mutation (wildtype) as well as the variants Alpha (B.1.1.7), Delta (AY.43) and Omicron (BA.1 and BA.5) was analyzed. Additionally, the neutralizing capacity of sera from ten patients was investigated (patient 1, 6, 9, 10, 24, 27, 33, 45, 48, 52) towards their equivalent clinical isolate that caused the SARS-CoV-2 breakthrough infection in comparison to wildtype isolate.
Neutralization assays were conducted as described previously (26 (link)). Briefly, two-fold serial dilutions of patient sera (1:20 to 1:2560) were pre-incubated with 100 TCID₅₀/50 µL SARS-CoV-2 for one hour at 37 °C. These mixtures were added to A549-AT cells and incubated for three days at 37 °C and 5% CO₂. Cell cultures were stained with 0.5% crystal violet (w/v) (Roth, Karlsruhe, Germany), solved in 20% (v/v) methanol (Merck, Darmstadt, Germany) and evaluated for CPEs by transmitted light microscopy. The highest serum dilution at which none of the triplicate cultures displayed CPEs was defined as the complete neutralization titer (NT₁₀₀).

Biofilm formation was analyzed according to O’Toole (2011) . Overnight grown cultures were washed in 1xPBS and OD₆₀₀ was adjusted to 0.1. Bacterial strains were cultivated in a microtiter plate in 100 μl modified M9 minimal medium with 0.5% casamino acids (Biozol Diagnostica Vertrieb GmbH, Germany) without shaking at 28°C. After incubation OD₆₀₀ was measured in the plate reader (SpectraMax iD3, Molecular Devices). After 24 h OD₆₀₀ was measured and unattached cells were dumped out of the plate. The plate was washed twice by submerging it in MilliQ water to further remove unattached cells. Hundred and twenty-five microliter of 0.1% crystal violet (Roth, Germany) solution was added to each well. After 15 min the plate was rinsed three times in MilliQ water and dried for 1.5 h before visual inspection of biofilm production. For quantification of the biofilm 125 μl of 30% acetic acid (Roth, Germany) was added to each well and incubated for 15 min at room temperature. The solution was transferred to a new microtiter plate and color intensity was quantified at the plate reader (SpectraMax iD3, Molecular Devices) with absorbance at 550 nm and 30% acetic acid as blank. Biofilm-forming Pseudomonas simiae WCS417 served as positive control and non-biofilm-producing Escherichia coli DH5α served as negative control. The experiment was repeated three times with 6–12 replicates per strain.

The neutralizing capacity of serum samples was determined by a standard end-point dilution assay, as previously described [16 (link)]. In brief, two-fold serial dilutions of patient sera (1:20 to 1:2560) were pre-incubated with 100 TCID₅₀/50µL SARS-CoV-2 for one hour at 37 °C. The mixtures were added to confluent Vero E6 cells cultured on 96-well microtiter plates supplemented with 10% FCS, penicillin (100 IU/mL) and streptomycin (100 µg/mL). To detect cytopathic effects (CPEs) via light microscopy, the cell cultures were stained with crystal violet (Roth, Karlsruhe, Germany) and dissolved in 20% methanol (Merck, Darmstadt, Germany) after 3 days of incubation. The neutralization titer was defined as the reciprocal of the highest serum dilution at which none of the triplicates showed signs of CPEs.

2 × 10⁵ cells were seeded on 50 µg mL⁻¹ fibronectin (Sigma–Aldrich) coated 96 well plates and allowed to adhere for 30 min at 37 °C under 5% CO₂. The optical density of the adhering cells was measured at 560 nm wavelength after having fixed the adherent cells with 2% glutaraldehyde (Roth) for 10 min, stained with 0.5 % crystal violet, and lysed with 10 % acetic acid (Roth). Quantification was obtained via optical density of crystal violet stained adherent cells.

PrestoBlue, fetal bovine serum (FBS), high glucose medium (4.5 g/L), and Dulbecco’s Modified Eagle’s medium (DMEM/F-12) were obtained from Life Technologies (Life Technologies, Carlsbad, CA, USA). Collagenase (type I) was purchased from Worthington Biochemical Corp. (Worthington Biochemical Corp., Lakewood, CA, USA). Paraformaldehyde (PFA), trypsin–EDTA, penicillin–streptomycin, and Tween^®20 were obtained from Sigma (Sigma Aldrich, St. Louis, MO, USA). Acetic acid and crystal violet were bought from Roth (Carl Roth GmbH, Karlsruhe, Germany). Basic fibroblast growth factor (bFGF) was obtained from PeproTech (PeproTech GmbH, Hamburg, Germany). Isopropyl alcohol Oil Red O from Merck (Merck KGaA, Darmstadt, Germany) was used. phosphate buffered saline (PBS) was bought from Biochrom (Biochrom GmbH, Berlin, Germany). Rosiglitazone was from LKT Laboratories Inc. (LKT Laboratories Inc., St Paul, MN, USA). The study protocol was approved by the regional ethics committee (Ethics Committee of the RWTH Aachen University Faculty of Medicine, Aachen, Germany; EK163/07).

Yeast extract, peptone, glucose, crystal violet and glacial acetic acid were obtained from Carl Roth GmbH (Karlsruhe, Germany), RPMI-1640 medium supplemented with L-glutamine was purchased at Thermo Fisher Scientific (Waltham, MA, USA). Statistical analysis was performed by two tailed unpaired student t-tests, where applicable. p values < 0.05 were considered significant. * denotes p < 0.05, ** < 0.01, *** < 0.001.

To assess the neutralizing antibody titers of sera from 28 KTX patients and 11 healthy controls, we used a standard endpoint dilution assay, as described previously [13 (link),17 (link),18 (link)]. From the respective sera, serial dilutions (1:20 to 1:2560) were incubated with 100 TCID₅₀ of SARS-CoV-2 D614G (wild type), alpha (B.1.1.7), delta (B.1.617.2) or omicron (BA.1) for one hour at 37 °C. Thereafter, the dilutions were added to confluent A549-AT cells [18 (link)] in 96-well microtiter plates. After three days of incubation, cells were stained with crystal violet (Roth, Karlsruhe, Germany) solved in 20% methanol (Merck, Darmstadt, Germany). Cells were evaluated for the presence of cytopathic effects (CPE) by light microscopy. The neutralizing titer was defined as the reciprocal of the highest serum dilution at which no CPE was observed in any of the three test wells. A549-AT cells overexpress carboxypeptidase angiotensin-I-converting enzyme 2 (ACE2) receptor and the cellular transmembrane protease serine 2 (TMPRSS2), enabling enhancement of CPE and high SARS-CoV-2 susceptibility. A549-AT cells were cultivated in minimum essential media (MEM), supplemented with 10% (v/v) FCS, penicillin (100 IU/mL), and streptomycin (100 µg/mL) at 37 °C in an atmosphere of 5% CO₂ (all Life Technologies Gibco, Darmstadt, Germany).