293fectin transfection reagent

We obtained the RBD-specific IgG S309 (PMID: 32422645) by transfecting Expi293 cells with plasmids that express S309 IgG heavy chain and light chain using the 293fectin transfection reagent (Thermo Fisher Scientific). On day 5 after transfection, the cell supernatant was clarified by centrifugation and filtered. The S309 antibody was purified from the supernatant by HiTrap rProtein A FF Column (Cytiva, Tokyo, Japan) using the AKTA go (Cytiva). We also utilized these heavy and light chain genes to produce an RBD-specific IgA expression plasmid, as described previously [14 (link)]. We obtained the recombinant anti-RBD-IgA antibody by transfecting this plasmid as the IgG antibody described above. The culture supernatant was purified using Peptide M/agarose (InvivoGen Inc., San Diego, CA, USA). These RBD-specific IgG and IgA antibodies served as a standard to measure RBD-specific antibodies. To assess the level of N-specific IgG and IgA antibodies, we purified the patient’s serum at one month post-infection and used it as a standard.

HeLa cells were seeded on a 35-mm dish (#150460, Thermo Fisher Scientific, Rockford, IL, USA) or a 6 well plate (#140675, Thermo Fisher Scientific). The plasmid expressing the CAHS1-mEGFP protein, the CAHS3-mEGFP, the CAHS8-mEGFP, the CAHS12-mEGFP, the LEAM-mEGFP, the MAHS-mEGFP and the mEGFP were transfected into the HeLa cells with 293fectin Transfection Reagent (#12347-019, Invitrogen, Waltham, MA, USA). After 24 h of transfection, the cells were loaded onto a microfluidic plate (M04S-03-5PK, Millipore, Burlington, MA, USA) according to the manufacturer’s instructions. The microfluidic plates were controlled by a CellASIC ONIX2 Microfluidic System (CAX2-S0000, Millipore). After 48 h of transfection, the culture medium was replaced with an imaging medium containing FluoroBrite DMEM (#A1896701, Thermo Fisher Scientific) supplemented with 1% GlutaMAX Supplement (#35050061, Thermo Fisher Scientific) and 0.1% bovine serum albumin. The imaging medium continuously flowed at 5 psi (“psi” is a unit of pressure) while heating at 37°C by a temperature-controllable manifold (CAX2-MXT20, Millipore).
For hyperosmotic shock experiments, 0.5 M sorbitol solution and 0.2 M sodium chloride solution were prepared as follows: 4.5 M sorbitol in DDW and 5 M sodium chloride in DDW were dissolved by the imaging medium, resulting in 0.5 M sorbitol solution and 0.2 M sodium chloride solution, respectively. The transfected cells seeded on microfluidic plates were time-lapse imaged under the imaging medium condition every 10 sec for 2 min. The imaging medium continuously flowed at 1 psi while heating at 37°C. Then, 0.5 M sorbitol solution or 0.2 M sodium chloride solution was added, and fluorescence images were acquired every 10 sec for 5 min. The hyperosmotic medium continuously flowed at 5 psi while heating at 37°C. After incubation, the hyperosmotic medium was replaced with the imaging medium, and the cells were observed every 10 sec for 10 min. The imaging medium continuously flowed at 5 psi while heating at 37°C.
Cells were imaged with an inverted microscope (IX83, Olympus/Evident, Tokyo, Japan) equipped with an sCMOS camera (ORCA-Fusion BT, Hamamatsu photonics, Hamamatsu, Japan), a spinning disk confocal unit (CSU-W1, Yokogawa Electric Corporation, Musashino, Japan), and diode lasers at a wavelength of 488 nm. An oil immersion objective lens (UPLXAPO100XO, N.A. 1.45, Olympus/Evident) was used. The excitation laser and fluorescence filter settings were as follows: excitation laser, 488 nm; dichroic mirror (DM), 405/488/561 nm; and emission filters, 500–550 nm. During the observation, the cells were incubated with microfluidic plates at 37°C. The microscope was controlled by MetaMorph software (Molecular Devices, San Jose, CA, USA).
Fluorescence imaging data were analyzed and quantified in Fiji/ImageJ (Schindelin et al., 2012 ). The background was subtracted by a constant value. If salt-and-pepper noise was not negligible, a median filter with 1-pixel was applied. To compensate for the misregistration caused during time-lapse imaging, the “StackReg function” was applied with the “Translation” option.

293F cells were cultured under FreeStyle™ 293 Expression Medium (Invitrogen). Transient transfection was performed by co-transfection of expression vectors encoding antibody heavy chain and light chain using 293fectin transfection reagent (Invitrogen), according to the manufacturer’s instructions. Four to five days later, supernatants from the transfected cells were harvested and tested for antibody secretion by ELISA. Briefly, 96-well plates (Nunc, Roskilde, Denmark) were coated with 1 μg/ml goat anti-human Fc gamma antibody in phosphate-buffered saline (PBS) for 16 hr at 4°C. After blocking for 1 hr with 0.4% BSA in PBS at room temperature, isolated supernatants were added in 1/3 sequential dilutions, and incubated for 1 hr at room temperature. Plates were subsequently washed three times and incubated with HRP-conjugated goat anti-human kappa-specific antibody for 1 hr at room temperature. After washing, plates were developed with OPT. The reaction was stopped with 2M H₂SO₄, and OD was measured at 490 nM.