Nunc maxisorp 96 well plate

Cell-free supernatants from HAF, transfected with either the EGFP or Piezo1 expression plasmid, were collected just before the start of nanoindentation experiments (3 days post-transfection) and stored at −20 °C. IL-6 was detected (in Nunc MaxiSorp 96-well plates (11530627, Invitrogen, Karlsruhe, Germany)) using a colorimetric assay based on the IL-6 Human Uncoated ELISA Kit (88-7066-88, Invitrogen) according to manufacturer’s instructions. Absorbance was measured with a Microplate Reader (Tecan Infinite 200) and analyzed with Magellan data analysis software.

Nunc MaxiSorp 96-well plates (Invitrogen, Carlsbad, CA, USA) were coated with rabbit anti-human C4d neoepitope-specific antibody [8 (link)]. After quenching with washing buffer (50 mM Tris-HCl, 0.15 M NaCl, 0.1% Tween, pH 7.5) supplemented with 3% fish gelatin (Norland Products, Cranbury, NJ, USA), patient or control plasma and pooled plasma from healthy volunteers (lacking C4d) supplemented with Escherichia coli-expressed C4d standard in serial dilutions were diluted to 4% in PBS with 0.02% Tween-20 and 0.02 M disodium ethylenediaminetetraacetate dihydrate and added to the plate. Detection was achieved using mouse anti-human C4d antibody (catalogue number 253; Quidel, San Diego, CA, USA) followed by horseradish peroxidase-conjugated goat anti-mouse secondary antibody (Dako, Carpinteria, CA, USA). Plates were developed using o-phenylenediamine dihydrochloride as a substrate, and absorbance was measured at 490 nm using a Varian Cary 50 microplate reader (Agilent Technologies, Santa Clara, CA, USA). The lowest detection limit was 5.6 μg/L. Values below the detection limit were set to 0.001 mg/L for statistical calculations. The inter-assay coefficient of variation was 16.7%, and the intra-assay coefficient of variation was 13.2%.

ELISA for serum and mucosal antibodies were performed as previously described [14 (link)]. Briefly, VLPs in PBS were incubated on Nunc MaxiSorp 96-well plates (Fisher Scientific) for 4 h at room temperature. The plates were blocked overnight at 4°C. All samples were serially diluted 2-fold and allowed to incubate at room temperature for 1 h. Plates were washed 5 times, followed by incubation with anti-guinea pig IgG-HRP (Southern Biotech, Birmingham, AL at 1:1000, anti-guinea-pig IgA (Creative Diagnostics, Shirley, NY) at 1:1000, anti-guinea pig IgG1 HRP (Antibodies Online) at 1:1000, or anti-guinea pig IgG2 RP (Antibodies Online) at 1:2000 for 1 h at room temperature. The plates were washed 5 times and developed with 1-step Ultra TMB according to the manufacturer’s protocol. End-point titers were reported as the reciprocal of the highest dilution that produced an OD of 0.1 above background and presented as Geometric Mean Titers. ositive control serum generated in guinea pigs against GI or GII.4 was included in each test run to confirm reproducibility.

Nunc MaxiSorp 96-well plates (Fisher Scientific, Richardson, TX, USA) were coated with 2.0 μg/mL of the recombinant KRT1-WT, KRT1-MU, or KRT1-DEL in 0.05 M carbonate/bicarbonate buffer (pH 9.6) by incubation at 4°C overnight. Plates were coated with 0.48 μg/mL of His-Tagged Human ATG8/GABARAPL1 protein (Sino Biological Inc., Beijing, CHN) as controls. The coated plates were blocked by incubation with 10% goat serum (Sigma-Aldrich) in PBS at 37°C for 2 h. The plates were then incubated with test serum diluted 1 : 50 in an incubation solution of 10% goat serum in PBS at 4°C overnight. After five washes with PBS, HRP-conjugated goat anti-human antibodies (Jackson Laboratories) diluted 1 : 5,000 were added, and samples were incubated at 37°C for 2 h. After five washes with PBS, 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonate) (ABTS, Shanghai Yuanye Biotechnology Co., Shanghai, CHN) was added to the plates, and the reaction was stopped after 30 minutes using 2 M sulfuric acid. Optical density was read using a Stat Fax-2100 microplate reader (Awareness Technology, Inc., Palm City, FL, USA) at 405 nm. Each sample was tested in triplicate, and a median value, from which the goat serum control value was subtracted, was used for analysis. Experiments on each sample were repeated to ensure reproducibility.