To evaluate T-cell proliferation, 96-well microtiter plates were coated overnight with anti-mouse CD3 antibody (100 μl/well of 1 μg/ml solution of Clone 145–2C11; BD Biosciences) or anti-rat CD3 antibody (100 μl/well of 1 μg/ml solution of Clone G4.18; BD Biosciences) and then washed. Spleen cell suspensions from Cohort 4 animals were separated from red blood cells (RBC) using Ficoll-Paque Plus (GE Healthcare) density gradients. The isolated cells were then re-suspended in complete RPMI 1640 medium (RPMI containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin; Gibco ThermoFisher Scientific) at 5 × 106 cells/ml. From this suspension, 100 μl of cells (5 × 105/well) were added to appropriate wells, and the plates incubated at 37 °C (in 5% CO2) for up to 72 h. Changes in total cell number, indicative of proliferation, were measured using a fluorescent nucleic acid stain assay (CyQuant Direct Cell Proliferation; ThermoFisher Scientific, Grand Island, NY) according to manufacturer instruct-tions. The fluorescent signal (485 nm excitation/535 nm emission), directly proportional to live cell number and thereby an index of proliferation, was measured using a Spectramax M2e spectrofluorometer and associated Softmax Pro software v5.0 (Molecular Devices).
Spectramax m2e spectrofluorometer
Manufactured by Molecular Devices
The SpectraMax M2e is a spectrofluorometer designed for high-performance fluorescence detection. It features a dual-monochromator system that allows for excitation and emission scanning. The instrument is capable of measuring fluorescence, luminescence, and absorbance in microplates, cuvettes, and other sample formats.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Ficoll paque plus, by GE Healthcare (2 mentions)
Softmax pro software v5, by Molecular Devices (2 mentions)
Clone 145 2c11, by BD (2 mentions)
Cyquant direct cell proliferation, by Thermo Fisher Scientific (2 mentions)
Clone g4, by BD (2 mentions)
3 protocols using spectramax m2e spectrofluorometer
1
Quantifying T-Cell Proliferation Assay
2
Quantifying T-Cell Proliferation Assay
3
Measuring Bacterial Cell Membrane Potential
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To analyze the effects of HTS-4 on the metabolic activity of bacterial cells, the membrane potential (MP) of the bacteria was measured according to the rhodamine 123 fluorescence method, as described by Comas and Vives-Rego [24] (link), with modifications. Bacterial cells were incubated in GAM medium at 37°C for 24 h. The cell solutions (∼1 × 107 CFU/mL) were added with different concentrations of HTS-4 (control, MIC, and MBC levels) and AGS (0.5 mg/mL) and incubated for 8 h. The suspensions were washed with PBS, and rhodamine 123 was then added to a final concentration of 2 μg/mL. After standing in the dark for 30 min, the samples were completely washed and resuspended in PBS. One-hundred microliters of the cell suspension was transferred into the wells of a 96-well microplate and placed in a SpectraMax M2e spectrofluorometer (Molecular Devices). The rhodamine 123 fluorescence was excited at 480 nm, and the emission wavelength was 530 nm. The data were expressed as the mean fluorescence intensity (MFI).
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