The specificity of purified scFv mAbs and sera from patients and controls were tested by ELISA as previous described.16 (link), 35 (link) Briefly, for testing the reactivity of PV mAbs to exogenous antigens and Dsg3, Stripwell Microplates (Corning, MA) were coated with 30ng/well of each antigen extract from Greer Labs or purified human recombinant Dsg3, and incubated overnight at 4°C. All ELISA experiments were conducted using Tris buffer saline (TBS) system. Washing buffer contained 10mM TBS, 0.05% Tween-20, and 5mM CaCl2. The blocking buffer consisted of washing buffer plus 1% BSA. All scFv mAbs, sera from patients and controls, secondary Abs were diluted in blocking buffer. The secondary Ab for scFv mAbs detection was HRP conjugated M2 anti-FLAG (Sigma). The bound serum antibodies were detected by HRP conjugated anti-human IgG1 (HP6001), IgG4 (HP6023), and IgM (SA-DA4) Abs, respectively (SouthernBiotech, Birmingham, AL). The plates were developed using the Pierce™ TMB Substrate (Pierce, Rockford, lL).
Stripwell microplate
Manufactured by Corning
Sourced in Morocco
Stripwell Microplates are a versatile laboratory tool designed for various applications. They feature a 96-well format with individual removable strips, allowing for flexible and efficient sample processing. The plates are made of high-quality materials and are suitable for numerous assays and experiments.
Automatically generated - may contain errors
Lab products found in correlation
Trehalose, by Merck Group (2 mentions)
Y1375, by Merck Group (2 mentions)
Anti flag m2, by Merck Group (1 mentions)
Breathe easy, by Merck Group (1 mentions)
Y1500, by Merck Group (1 mentions)
Tetramethylbenzidine substrate, by Thermo Fisher Scientific (1 mentions)
Crystal violet, by Merck Group (1 mentions)
6 protocols using stripwell microplate
1
ELISA Evaluation of scFv mAbs and Sera
2
Measuring Cell Stiffness via Magnetic Twisting
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Cell stiffness was measured using optical magnetic twisting cytometry41 (link). ASM cells were cultured to confluence in collagen-coated 96-well plate (Stripwell Microplates, Corning, NY). After overnight serum starvation, cells were incubated with ferrimagnetic beads (4.5 μm in diameter, produced in house, and coated with poly-L-lysine) for 20 min to allow beads to attach to cell surfaces74 (link). The beads were then magnetized with a strong magnetic pulse in the horizontal direction and twisted with a much weaker oscillatory magnetic field (0.77 Hz) in the vertical direction. The ratio of magnetic torque to bead motion41 (link),74 (link) was used to determine cell stiffness; values are expressed in arbitrary units (a.u). To determine relative changes in cell stiffness after drug treatment, the average of stiffness normalized to baseline was calculated.
3
Air-Drying Budding Yeast for Spaceflight
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The method to air-dry budding yeast cells for the BioSentinel mission has been described previously (Santa Maria et al., 2020 (link)). Briefly, yeast from frozen glycerol stocks were grown on yeast extract-peptone-dextrose (YPD) agar (Y1500; Sigma-Aldrich) for 2–3 days at 30°C. Samples from freshly grown patches were then inoculated into 5 mL of liquid YPD (Y1375; Sigma-Aldrich) and grown on a rotating mixer at ambient temperature (∼23°C) for 7 days. Liquid cultures were pelleted and washed with sterile water, and the cell density was determined by hemacytometer counting. Cell suspensions were prepared to a final density of 1 × 107 cells in 1 mL of 10% trehalose (T0167; Sigma-Aldrich). Ten microliter aliquots of the 1 × 107 cell suspension (containing ∼105 cells) were gently dispensed into the bottom edge of the wells of 96-well Stripwell™ microplates (9102; Costar®) or onto the sidewall of each microfluidic card well (Padgen et al., 2021 (link)).
Plates were sealed with Breathe-Easy® gas permeable sealing membrane (Z380059; Sigma-Aldrich), and the cells were air dried in a 23°C incubator, 20–30% relative humidity, for 7 days. Yeast cells in fluidic cards were air dried inside sterile boxes at the same conditions before card sealing using a pneumatic press (Padgen et al., 2021 (link)).
Plates were sealed with Breathe-Easy® gas permeable sealing membrane (Z380059; Sigma-Aldrich), and the cells were air dried in a 23°C incubator, 20–30% relative humidity, for 7 days. Yeast cells in fluidic cards were air dried inside sterile boxes at the same conditions before card sealing using a pneumatic press (Padgen et al., 2021 (link)).
4
Desiccation Resistance Assay for Yeast Strains
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Wild type and rad51Δ diploid strains from −80°C glycerol stocks were plated on yeast extract - peptone - dextrose (YPD) agar (Sigma-Aldrich; Y1500) and incubated at 30°C for 2–3 days. Samples from freshly grown patches were then used to inoculate 5-mL YPD liquid cultures (Sigma-Aldrich; Y1375), which were grown on a rotating mixer at ambient room temperature (∼23°C) for 7 days. Liquid cultures were pelleted and washed with 1 mL sterile deionized water and then resuspended in 1 mL sterile deionized water. Cell density was determined by hemocytometer counting, and suspensions were diluted to a final density of 1 × 107 cells/mL in 10% (w/v) trehalose (Sigma-Aldrich; T0167). Ten microliters of trehalose suspensions (containing ∼105 cells) were gently dispensed into the bottom edge of the wells of 96-well Stripwell™ microplates (Costar®; 9102). Undesiccated controls were plated on YPD agar (Sigma-Aldrich; Y1500) for colony counting.
5
SARS-CoV-2 S Protein Binding Assay
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Full-length His-tagged SARS-CoV-2 S protein (Sino Biological Inc., 40589-V08B1) (5 μg/ml) was coated on a Costar StripwellTM Microplate at 4 °C overnight (50 μl/well). The plates were blocked with 5% non-fat milk in PBS for 2 h at 37 °C. Subsequently, the plates were washed three times with PBS containing 0.1% v/v Tween-20 (PBS-T), followed by incubation with 10-fold serially diluted serum from mice in PBS-T for 1 h at 37 °C. After washing three times with PBS-T, A 1:10,000 dilution of HRP-conjugated goat anti-mouse IgG antibody or HRP-conjugated goat anti-mouse IgM antibody was incubated in each well for 1 h at 37 °C. The plates were then washed four times with PBS-T, followed by adding 100 μl of tetramethylbenzidine substrate (Invitrogen) at room temperature in the dark. After 10 min of incubation, the reaction was stopped with 100 μl 2 M H2SO4 solution. The absorbance of each well was measured at 450 nm.
6
Quantifying Biofilm Formation Assay
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The biofilm formation were measured by detecting the ability of the cells to attach to the wells of polystyrene StripwellTM Microplate (1 X 8 Flat Bottom; Corning incorporated, Costar, Code#: 42592) as previously described with minor modifications [7 (link),46 (link)]. Briefly, an overnight PB culture was diluted to a final OD600 of 0.05 in fresh PB medium and dispensed 100 μL into per well. The plates were incubated under static conditions for 72 h at 37 °C. In order to measure the degree of attachment, non-adhered cells were removed, and the biofilms rinsed with distilled water. Biofilms were stained by 150 μL of 1% crystal violet (Cat#: 3603, Sigma-Aldrich) for 15 min. Photos were taken and crystal violet was solubilized in 150 μL of 30% acetic acids, the extent of biofilm formation was quantified by measuring the OD595 of the resulting solution. The statistics comparison was done by Student’s two-tailed t-test in GraphPad Prism (version 7.0).
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