Anti-CD19 CAR T cells were co-cultured with target cell lines with designated E:T ratios (1:1 and 5:1) for 4 hours (Table 1 ). CD19 positive Raji cells and CD19 negative K562 cells were used as positive and negative control cells respectively. Target cells were subsequently stained with PE-conjugated mouse anti-human CD19 antibody (BD Biosciences, San Jose, CA) and analyzed by FACSCanto flow cytometry. The target cells were suspended in RPMI-1640 containing 10% FBS, labeled with calcein AM (BD Biosciences, San Jose, CA), and plated onto 96-well flat-bottom plates (Costar, Corning, NY). UWC19 cells, suspended in RPMI-1640 containing 10% FBS, were then added at various E:T ratios and co-cultured with the target cells for 4 h. Following this, the cells were stained with propidium iodide (Sigma-Aldrich, St. Louis, MO), and the cytotoxicity was assessed by flow cytometry on a FACSCanto (Becton Dickinson) instrument enumerating the number of viable target cells (calcein AM-positive, propidium-iodide negative, and light scattering properties of viable cells).
96 well flat bottom plate
Manufactured by Corning
Sourced in United States, Germany, Switzerland
The 96-well flat-bottom plates are a type of laboratory equipment used in various scientific and medical applications. These plates consist of a grid of 96 individual wells, each with a flat bottom. The plates are commonly used for a variety of assays, sample preparation, and high-throughput screening procedures.
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Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (9 mentions)
Celltiter 96 aqueous one solution cell proliferation assay, by Promega (7 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Calcein am, by BD (4 mentions)
Phorbol myristate acetate (pma), by Merck Group (4 mentions)
Prism 7, by GraphPad (4 mentions)
Ficoll paque, by GE Healthcare (4 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (4 mentions)
Synergy h1, by Agilent Technologies (4 mentions)
Glutamine, by Thermo Fisher Scientific (4 mentions)
Il 21, by Thermo Fisher Scientific (3 mentions)
Rpmi 1640, by Corning (3 mentions)
Fluostar omega plate reader, by BMG Labtech (3 mentions)
Synergy 2, by Agilent Technologies (3 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (3 mentions)
Elisa, by BD (3 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (3 mentions)
Lipopolysaccharides (lps), by Merck Group (3 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Microplate reader, by Tecan (3 mentions)
294 protocols using 96 well flat bottom plate
1
Cytotoxicity Assay for Anti-CD19 CAR T Cells
2
Cytotoxicity Assay for NK Cells
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The target cells were suspended in RPMI-1640 containing 10% FBS, labeled with calcein AM (BD Biosciences, San Jose, CA), and plated onto 96-well flat-bottom plates (Costar, Corning, NY). The NK cells, suspended in RPMI-1640 containing 10% FBS, were then added at various E:T ratios and co-cultured with the target cells for 4 h. Following this, the cells were stained with propidium iodide (Sigma-Aldrich, St. Louis, MO), and the cytotoxicity was assessed by flow cytometry on a FACSCalibur (Becton Dickinson) instrument enumerating the number of viable target cells (calcein AM-positive, propidium-iodide negative, and light scattering properties of viable cells) [3] (link), [26] (link).
3
Influenza Virus Inhibition Assay
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96-well flat-bottom plates (Costar) were seeded with 1.5 x 104 A549 cells/well and incubated overnight at 37°C/5%CO2. Clopidogrel and Triamterene were prepared in DMSO to a stock concentration of 10 mM. Drugs plates were prepared using the D300 BioPrinter digital drug dispenser (HP, Palo Alto, CA) and dispensed into 96-well plates containing virus infection media containing MEM supplemented with 0.3% BSA and TPCK-treated trypsin (1 ug/ml) (Worthington, Lakewood, New Jersey) to the final concentrations 500, 200, 150, or 100 uM. A/WSN/33 virus (MOI = 0.01) was prepared in infection media. A549 cell plates were washed 2x with PBS. Equal volumes of infection and drug dilutions were transferred to the A549 cells (excluding control wells) to infect cells (MOI = 0.01) and achieve final concentrations of 250, 100, 75, or 50 uM of the drug. The wells were normalized to 1% DMSO. The plates were incubated for 12h or 24h at 37°C/5% CO2. Following incubation, the supernatants were removed, the plates were washed 2x with PBS, and the cells fixed with acetone:methanol (20:80; Sigma). Leptomycin B (LMB) (Sigma, St. Louis, MO) was used as the positive control for inhibition of influenza replication and was administer on cells 2h prior to infection [58 (link), 59 (link)].
4
Anti-FZ Antibody ELISA Assay
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Anti-FZ IgG1, IgG2a, and IgA antibody ELISAs were performed using CoStar 96-well flat bottom plates (Washington, DC) similar to as previously described13 (link),24 (link). Briefly, wells were coated with 1 µg/ml FZ or recombinant standards (IgG1, IgG2a (Millipore Sigma) or IgA (Southern Biotech, Birmingham, AL)), incubated with sample dilutions, and detected with AKP-conjugated anti-mouse IgG1, IgG2a (BD Biosciences, Franklin Lakes, NJ) or HRP-conjugated anti-mouse IgA (Millipore Sigma). Results reported as ELISA units/ml (EU/ml) are averaged interpolated values around the standard curve midpoint. Cytokines were analyzed with thawed supernatants using Mouse CD8 T-cell Panel (Millipore Sigma) with a Bioplex 200 array reader (Bio-Rad, Hercules, CA).
5
Macrophage Cytokine Response to LPS
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RAW 264.7 macrophages were cultured in RPMI-1640 medium supplemented with 10% foetal bovine serum. The cells (1 × 104) were plated in 96-well flat-bottom plates (Corning Costar, Corning, NY) and incubated in the presence of E. coli LPS or Bacteroides LPS for 12, 24, 48, and 72 h at 37 °C with 5% CO2. The cytokine levels were analysed using the Cytometric Bead Array Kit (BD Biosciences, San Diego, CA, USA), according to the manufacturer’s instructions.
6
Mixed Lymphocyte Reaction with Cyclosporine
7
Cell Viability Assay via MTT
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In 96-well flat bottom plates (Costar, Corning, NY), cells were seeded at a density of 5000 cells/50ul. Cells were incubated overnight and the next day drug treatments were administered up to a final volume of 100ul. For MTT analysis, 42 μl of a 5-mg/ml solution of MTT tetrazolium substrate (Sigma) in phosphate-buffered saline was added and incubated for up to 2 hours at 37 °C. The resulting violet formazan precipitate was solubilized by the addition of 84 μl of 0.01 M HCl in 10% SDS (Sigma-Aldrich, St. Louis, MO) solution at 37 °C overnight. Plates were analyzed on a microplate reader at 570 nm (Synergy Mx Monochromator-Based Multi-Mode Microplate Reader, Biotek Instruments, Winooski, VT).
8
ZIKV and DENV Antibody ELISA
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Enzyme-linked immune sorbent assay (ELISA) was used to determine the antibody response levels elicited by E.coli and S2 expressed ZIKV E80 proteins using serum samples obtained from immunized mice, and the cross reactivity of ZIKV immune sera with DENV E80 protein. Briefly, 96-well flat-bottom plates (Costar) were coated overnight with 100μl of diluted ZIKV E80 protein or DENV-3 E80 protein, at a final concentration of 0.5μg/ml. The plates were decanted and then blocked with 5% no-fat milk in PBS containing 0.05% Tween 20 (PBST) for 2 hours at 37°C. Two fold diluted inactivated serum samples were then added in duplicate wells and incubated for 1 hour at 37°C. Horseradish peroxidase (HRP)-conjugated anti-human antibody (Invitrogen) was then added and incubated for 1 hour. Color development was performed using TMB single solution (Life technology), followed by the colorimetric analysis at 450 nm in a 96-well plate reader (Thermo Fisher Scientific).
9
MM Cell Culture and Drug Treatment
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We cultured the MM cells in RPMI-1640 (Gibco, Darmstadt, Germany) supplemented with 10% fetal bovine serum at 1 × 10e5 cells/well in 96-well flat-bottom plates (Costar, Washington, DC). We reconstituted ricolinostat, ACY-241 and WT-161 in dimethylsulfoxide and added them to the medium at final concentrations of 1, 5, and 10 µM. We likewise reconstituted ATRA and Panobinostat in dimethylsulfoxide and added these compounds to the medium at final concentrations of 10 nM.
10
Quantifying Acetate Levels in Microalgae
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Acetate remaining in culture media was quantified using the Acetate Colorimetric Assay Kit (BioVision, Milpitas, CA, USA). Both N replete and N-depleted TAP medium for wild-type and tab2 mutant cells contained 17.5 mM acetate prior to culturing at 0 h. One milliliter of the culture was withdrawn from the flask at each time point. The supernatant was filtered through a 0.45 μm filter membrane after centrifugation at 14,000×g for 5 min and then was stored at −20 °C. All samples were analyzed using 96-well flat bottom plates (Costar, Corning, NY, USA) following the kit protocol by measuring the absorption maximum at 450 nm in a SPECTRAmax PLUS microplate spectrophotometer system (Sunnyvale, CA, USA).
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