Anti-HER2 antibody glycoforms were tested in a robust ADCC assay (PMID 25086226)23 (link) against the human breast carcinoma cell line SKBR3 (HER2+, ATCC). In brief, SKBR3 cells were plated 24 h prior to the assay in opaque 96-well flat bottom plates (Costar) at 104 cells/well. On the day of the assay, each glycoform was prepared at 5 μg/mL and 3-fold serial dilutions were made in a separate 96-well plate. Each was then added, in duplicate, to the SKBR3 cells and incubated for 30 min. The effector cells (Jurkat cell line expressing the F allele of FcγRIIIA, Promega) were thawed per the manufacturer’s recommendations and added to the plate at a concentration of 7.5 × 104 cells/well. The plates were then incubated for 6 h at 37 °C (5% CO2). Following incubation, the plates were equilibrated to room temperature for 15 min prior to the addition of Bio-Glo Luciferase Assay Reagent (Promega). Cells were allowed to develop at room temperature for 15 min prior to reading on a SpectraMax Plus spectrophotometer (Molecular Devices). Background luminescence from negative control samples was subtracted, and signal was expressed as relative luciferase units.
Opaque 96 well flat bottom plates
Manufactured by Corning
Opaque 96-well flat bottom plates are laboratory equipment designed for various applications. These plates feature 96 wells with flat bottoms, and the wells are opaque in nature. The core function of these plates is to provide a standardized platform for conducting experiments, assays, or other laboratory procedures that require a multi-well format.
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2 protocols using opaque 96 well flat bottom plates
1
ADCC Assay for Anti-HER2 Antibody Glycoforms
2
SARS-CoV-2 Microneutralization Assay
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Vero E6 cells (ATCC CRL-1586) were seeded at a density of 2.5 × 104 cells per well in opaque 96 well flat-bottom plates (Costar) in complete DMEM (supplemented with 10% FBS, 1% L-glutamine, 100 U/mL penicillin-streptomycin). Twenty-four hours later, serum (resolved and RT-PCR-negative subjects) was inactivated by incubating at 56 °C for 30 min, then diluted 1:10 in low serum DMEM (supplemented with 2% FBS, 1% L-glutamine, 100 U/mL penicillin-streptomycin), followed by a 1:2 dilution series in 96 well U-bottom plates resulting in a final volume of 55 µL diluted serum per well. An equal volume of SARS-CoV-2/SB3-TYAGNC consisting of 330 PFU per well was then added to the diluted serum and the serum-virus mixture was incubated at 37 °C for 1 h. Next, the Vero E6 culture media was then replaced with 100 μL of the serum–virus mixture and was incubated at 37 °C for 72 h. The plates were read by removing 50 μL of culture supernatant and adding 50 µL of CellTiter-Glo 2.0 Reagent (Promega, G9243) to each well. The plates were then shaken at 282 cpm at 3 mm diameter for 2 min, incubated for 5 min at room temperature and luminescence was read using a BioTek Synergy H1 microplate reader with a gain of 135 and an integration time of 1 s. Results are expressed as geometric microneutralization titers at 50% (MNT50).
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