Passive lysis 5 buffer

FXR reporter cells were seeded at a density of 5 × 10⁴ cells/well (96-well plates). Twenty-four hours after seeding, cells were incubated with bacterial suspensions, culture supernatants, dimethyl sulfoxide (DMSO, 0.1 % v/v) or GW4064 (10 μM) respectively for 24 h. Then, cell supernatants were removed and cells were washed twice with phosphate-buffered saline (PBS) and lysed by adding 20 μl of passive lysis 5 × buffer (Promega) with gentle rocking for 20 min. Luciferase activity was measured by the administration of luciferase assay reagent (Promega) using a GloMax® 96 Microplate Luminometer (Promega). The ratio of treatment over control was used to determine the fold activation.

Cells were lysed in passive lysis 5× buffer (Promega, Madison, Wisconsin, USA), diluted in water, and contained complete ethylenediaminetetraacetic acid–free protease inhibitor cocktail (Roche Applied Science, Basel, Switzerland) as recommended by the manufacturer. Protein concentration was measured using a Bradford protein assay and a Sunrise 96‐well plate reader (Tecan Group Ltd, Männedorf, Switzerland). The protein was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, blotted onto a polyvinylidene difluoride membrane, and probed for expression of the V5 epitope and α‐actin (see Supporting Information Section S3).

SARS-CoV-2 pseudovirus was manufactured by Sinocelltech (Beijing, China). Huh-7 cell was obtained from Chinese Culture Tissue Collection Center (CCTCC, China). Luciferase Assay System (E1501) and Passive Lysis 5× Buffer (E1941) were purchased from Promega (Madison, WI, USA). For the quantitative measurement of neutralization antibodies, the SARS-CoV-2 pseudovirus PsV-Luc-Spike carrying the firefly luciferase gene was used. Mutations in S-ECD of each variant pseudoviruses are shown in Table 1. Briefly, vaccine-immunized serum samples (heat-inactivated at 56 °C for 30 min) were serially diluted, incubated with 200 TCID₅₀/well pseudovirus (1 h at 37 °C, in a 5% CO₂ incubator), and co-cultured with 2 × 10⁴ Huh-7 cells for 20 h. Relative light unit (RLU) was measured to evaluate luciferase activity (CentroXS³ LB 960 Microplate Luminometer). The calculation formula for the inhibition rate of the pseudovirus entry is: Inhibition (%) = (Postive RLU-Sample RLU)/(Postive RLU-Negative RLU) × 100%. The neutralizing antibody titer (50% inhibitory dilution, NAT₅₀) is defined as the serum dilution at which the RLUs were reduced by 50% when compared with the positive control wells. Positive neutralizing antibody (NAb) was determined as greater than 50% inhibition, and the dose required to achieve this effect in 50% of the animals (ED₅₀) was calculated.

Cell lysis was performed with a Passive Lysis 5× Buffer (Promega, Cat. no: E1941). 50 μL of the buffer and 150 μL of distilled water was added to 50 μL of the cell sample to obtain a fivefold dilution of the buffer. The lysis was kept under stirring for 20 min, after which the samples were centrifuged and the supernatant was removed.
Total protein concentration was measured by the Bradford method. A total of 20 µL of a sample was added to 200 µL of reagent (0.01% Coomassie brilliant blue G-250, 4.7% ethanol, 8.5% phosphoric acid in distilled water). Detection was carried out at 590 nm within a 15-min time period.

The culture medium was removed from the wells and the plate was washed with PBS. The Passive Lysis 5× Buffer (Promega E1941) was used to perform cell lysis. A total of 70 µL of 5× diluted buffer was added to each well. The lysis process was carried out for 20 min with continuous moderate agitation on a plate shaker (StatFax 2200 Avarness Technology Inc., Palm City, FL, USA) at room temperature until the cells detached from the plate surface. Lysates were pipetted off and centrifuged, while the obtained supernatant was placed in clean, labelled Eppendorf type tubes and stored in the freezer (−20 °C) for further analysis.