Clariostar

For protein extraction, we used freeze-dried biomass obtained after 14 days of cultivation. Basically, for solubilization, we left 5 mg microalgae powder suspended in 50 μL of 1 M NaOH for 10 min in a water bath at 80 °C. Then, we added 450 μL of distilled water, and the resulting solution was centrifuged for 30 min at 12,000× g (in a Universal 320 R centrifuge, Hettich, Tuttlingen, Germany), and the supernatant was moved to another tube. The above procedure was repeated twice, and the last extract was combined. The protein content was determined using two methods, Bradford and Biuret. For the Bradford method, we used bovine serum albumin (BSA) as standard in a concentration series of 0–10 µg/mL [95 (link)]. Briefly, over 200 µL of a sample, we added 50 µL of Bradford Reagent (Bradford Reagent, Sigma, Merck Group, Darmstadt, Germany), incubated for 5 min, and read the absorbance in a 96 well plate reader (CLARIOStar, BMG Labtech, Ortenberg, Germany) at a wavelength λ of 595 nm. For Biuret, the BSA standard curve was 0–10 mg/mL. Briefly, we added 200 µL of Biuret reagent to 40 µL of the sample; we incubated it in the dark at room temperature for 30 min, and then the absorbance (CLARIOStar, BMG Labtech, Ortenberg, Germany) was read at the wavelength λ = 555 nm.

Myxoma virus titers were determined by standard plaque assay on RK13 cells, as described in Camus-Bouclainville et al. (30 (link)). Enterovirus D68 and influenza virus titers were determined by the tissue culture infectious dose 50 (TCID₅₀) method, as described in Soubies et al. (38 (link)). We measured replication of luciferase expressing virus 24 h post-infection by lysing cells and measuring light emission on a Clariostar (BMG Labtech) plate reader using the Luciferase assay System kit (Promega) according to the manufacturer's instructions. We measured replication of TMEV expressing the fluorescent protein Cherry by measuring fluorescence using a Clariostar (BMG Labtech) plate reader. hRSV expressing Cherry was detected in paraformaldehyde fixed HEp-2 cells by immunofluorescence and imaged using a confocal microscope. Replication of hAdV and hCMV expressing AnchOR3 protein was quantified by measuring GFP foci using automated microscopy, as described in Komatsu et al. (27 (link)) and Mariamé et al. (31 (link)).

Flp-In T-REx 293 cell lines able to express hM₁-mEGFP were grown to 100,000 cells/well in 96-well solid black bottom plates (Greiner Bio-One) precoated with 0.1 mg·ml⁻¹ poly-d-lysine. Cells were treated with 100 ng·ml⁻¹ doxycycline to induce the expression of hM₁-mEGFP. After 24-h induction, cells were washed three times in Hanks' balanced salt solution buffer. 100 μl of Hanks' balanced salt solution was added to each well, and the plates were read using a CLARIOstar fluorescence plate reader (BMG Labtechnologies). Specifically, cells were excited at 462 nm, and the emission spectrum between 500 and 600 nm was collected at 5-nm intervals. The same process was repeated after the addition to each well of 100 μl of Hanks' balanced salt solution supplemented with the vehicle or the appropriate muscarinic receptor antagonist.

HCV neutralization assays were performed as described.^[28] Briefly, HEK293T cells were cotransfected in a 1:1 (w/w) ratio of pE1E2H77c (gt‐1a; Genbank AF011751) or pUKN3A1.9 (gt‐3a; Genbank AY734985) and pNL4–3.LUC.R‐E‐ to produce HCVpp. Alternatively, infectious HCV cell culture (HCVcc)‐derived genotype 3a (S52; Genbank EU204645), and 5a (SA13; Genbank FJ393024) were produced by transfecting Huh7.5 cells with in vitro–transcribed RNA by electroporation,^[26] with the modification that luciferase activity was measured in a microplate reader (ClarioSTAR; BMG Labtechnologies). Mean percentage entry was calculated as (relative light units [RLU] plasma + virus)/(RLU medium+virus) × 100. Percentage entry was plotted against the reciprocal dilution of plasma in GraphPad Prism software (version 9; GraphPad Software) and curves fitted with a one‐site specific binding Hill plot. The reciprocal dilution of plasma required to prevent 50% virus entry was calculated from the nonlinear regression line (ID₅₀). The lowest amount of nAb detectable was a titer of 40. Samples that did not reach 50% neutralization were assigned an arbitrary value of 20.

HCV neutralization assays were performed as described previously (53 (link)). Briefly, HEK293T cells were co-transfected in a 1:1 (w/w) ratio of pE1E2H77c and pNL4–3.LUC.R-E- to produce HCV H77pp (54 (link), 55 (link)). 1:40 dilutions of guinea pig sera were added to H77pp and incubated for 1 h at 37 °C before addition to Huh7.5 cells. After incubation for 4 h, the inocula were removed, and cells were incubated in fresh media for 72 h. Following lysis in cell culture lysis buffer (Promega, Madison WI), luciferase activity in clarified lysates was measured by using a luciferase substrate (Promega) on a CLARIOstar microplate reader fitted with luminescence optics (BMG Lab Technologies). Infectious cell culture–derived genotype 2a (J6), 3a (S52), and 5a (SA13) HCVcc were produced by transfecting Huh7.5s with in vitro–transcribed RNA by electroporation as described previously (25 (link)). NAb assays were performed by mixing HCVcc with 1:40 dilutions of guinea pig sera as described above with incubation for 42 h after removal of the inocula. Luciferase activity in cell lysates was measured using Renilla luciferase substrate (Promega).

Assay uses the PiColorLock Gold Phosphate Detection System from Novus Biologicals following the manufacturer’s recommended protocol. Briefly, assays were carried out in 96-well clear flat-bottomed plates, with absorbance measurements taken at a wavelength of 630 nm in a CLARIOstar multimode plate reader (BMG Labtech). Assay buffer: 50 mM Tris-HCl pH 7.5, 50 mM NaCl, 2 mM MgCl₂, 0.05% v/v Tween-20, 0.5 mM TCEP.
165 μl of BLM-HD and ssDNA-20mer (at a concentration of 2.4 nM and 121 nM, respectively) was pre-incubated with 10 μl of compound (2 mM stock dissolved in 100 % v/v DMSO, over a range of final concentrations up to 100 µM) for a period of 15 min at room temperature. Next, 25 µl of Mg-ATP substrate (at 16 mM) was added. After 20 min, reactions were stopped by the addition of 50 μl Gold mix (a 100:1 ratio of PiColorLock:Accelerator reagents). After 2 min, 20 μl of stabiliser solution was added to each well. After a further 30 min absorbance measurements were taken.
Assay conditions: 2 nM BLM-HD, 100 nM ssDNA-20mer and 2 mM Mg-ATP in a reaction volume of 200 µl over a 20-min incubation period.