Fluostar galaxy microplate reader

The in vitro caspase-3 activity was determined by fluorometric measurement of the kinetics of 7-amino-4-trifluoromethylcoumarine (AFC) release from the fluorogenic substrate Ac-DEVD-AFC in the presence of cell lysates. The method was described in detail previously [23] . Following cell incubation with 20 μM IPA-3, the cells (3×10⁶) were washed, lysed and aliquots of cytosolic proteins were incubated with Ac-DEVD-AFC for 30 min at 37°C. Linear increase in fluorescence intensity at 520 nm was monitored using Fluostar Galaxy microplate reader (BMG Labtechnologies, Germany) and the slope was used as a relative value of caspase-3 activity.

FRT cells transfected with CFTR were plated in a black 96-well plate with clear bottom (Costar, Corning, NY, USA) at a density of 2 × 10⁴ cells/well and incubated until confluent. The cells were washed three times with PBS followed by the addition of FSK (100 nM per well) and a further incubation of 5 min. After that, kobusin or eudesmin was added to each well at different concentrations and the cells were incubated for another 15 min. YFP fluorescence data were recorded using a FLUOstar Galaxy microplate reader (BMG Lab Technologies, Inc.) equipped with HQ500/20X (500 ± 10 nm) excitation, HQ 535/30M (535 ± 15 nm) emission filters (Chroma Technology Corp.) and syringe pumps. Iodide influx rates (d[I–]/dt) were computed as described by Kristidis et al. (1992) (link).

Membrane permeabilization was assessed by monitoring the hydrolysis of ortho-nitrophenyl β-D-galactopyranoside (ONPG) by the β-galactosidase enzyme, using the S. aureus ST1065 strain, specifically designed for membrane permeabilization assays, through detection of cytoplasmic β-galactosidase activity leakage [23 (link)]. Bacteria were grown in LB to an absorbance of 0.5 at 600 nm, pelleted by centrifugation, and the pellets were washed twice with PBS. Pellets were then resuspended in 10% LB in PBS to an absorbance of 0.5 at 600 nm. Aliquots (15 μL) of bacterial suspension were mixed with 2.5 mM ONPG and incubated with various concentrations of peptide in a 96-well plate. The hydrolysis of ONPG was monitored by measuring the absorbance at 420 nm of released o-nitrophenol with a Fluostar Galaxy microplate reader (BMG LabTech, France) [17 (link)].

HA-GLUT4 levels on the PM were determined as described previously (Hoehn et al., 2009 (link); Govers et al., 2004 (link)). L6 myotubes stably overexpressing HA-Glut4 were washed twice with warm PBS and serum-starved for 2 hr (in DMEM/0.2% BSA/GlutaMAX/with 220 mM bicarbonate [pH 7.4] at 37°C, 10% CO₂). Cells were then stimulated with insulin for 20 min, after which the cells were placed on ice and washed three times with ice-cold PBS. Cells were blocked with ice-cold 10% horse serum in PBS for 20 min, fixed with 4% paraformaldehyde (PFA) for 5 min on ice and 20 min at room temperature. PFA was quenched with 50 mM glycine in PBS for 5 min at room temperature. We measured the accessibility of the HA epitope to an anti-HA antibody (Covance, 16B12) for 1 hr at room temperature. Cells were then incubated with 20 mg/mL goat anti-mouse Alexa-488-conjugated secondary antibody (Thermo Fisher Scientific) for 45 min at room temperature. The determination of total HA-GLUT4 was performed in a separate set of cells following permeabilization with 0.01% saponin (w/v) and anti-HA staining (as above). Each experimental treatment group had its own total HA-GLUT4. A FLuostar Galaxy microplate reader (BMG LABTECH) was used to measure fluorescence (excitation 485 nm/emission 520 nm). Surface HA-GLUT4 was expressed as a fold over control insulin condition.

Permeabilization of the E. coli ML35p membrane by both peptides was assayed by measuring the β-galactosidase activity with the chromogenic substrate o-nitrophenyl-β-D-galactopyranoside (ONPG). Bacteria were grown in 10% Mueller–Hinton broth with 50 µg/mL ampicillin, washed twice with the test buffer (10 mM phosphate, 100 mM NaCl at pH 7.4 and 300 µg/mL Mueller–Hinton broth), and diluted in the test buffer to an absorbance of 0.6 at 630 nm (mid-log phase). 50 µL aliquots of bacterial suspension were mixed with 25 µL pf the peptide at various concentrations and 25 µL ONPG (2 mM, i.e., 0.5 mM final) in a 96-well plate. The hydrolysis of ONPG was monitored by measuring the absorbance of released o-nitrophenol at 412 nm with a Fluostar Galaxy microplate reader (BMG LabTech, France) [8 (link)]. Data shown are the means of 3 independent experiments realized in duplicate.

HA-GLUT4 levels on the plasma membrane were determined as previously described^{9 (link),84 (link)}. L6-myotubes stably overexpressing HA-Glut4 were washed twice with warm PBS and serum-starved for two hours (in DMEM/0.2% BSA/GlutaMAX/with 220 mM bicarbonate (pH 7.4) at 37 °C, 10 % CO2). Cells were then stimulated with insulin for twenty minutes, after which the cells were placed on ice and washed three times with ice cold PBS. Cells were blocked with ice cold 10 % horse serum in PBS for 20 min, fixed with 4 % paraformaldehyde (PFA) for 5 min on ice and 20 min at room temperature. PFA was quenched with 50 mM glycine in PBS for 5 min at room temperature. We measured the accessibility of the HA epitope to an anti-HA antibody (Covance, 16B12) for 1 h at room temperature. Cells were then incubated with 20 mg/mL goat anti-mouse Alexa-488-conjugated secondary antibody (Thermo Fisher Scientific) for 45 min at room temperature. The determination of total HA-GLUT4 was performed in a separate set of cells following permeabilization with 0.01% saponin (w/v) and anti-HA staining (as above). Each experimental treatment group had its own total HA-GLUT4. A FLuostar Galaxy microplate reader (BMG LABTECH) was used to measure fluorescence (excitation 485 nm/emission 520 nm). Surface HA-GLUT4 was expressed as a fold over control insulin condition.