Spark m10

Quantification of surface-available chitosan was performed as previously described (Muzzarelli, 1998 (link); Jøraholmen et al., 2015 (link)). In short, glycine buffer (250 ml, pH 3.2) was prepared in distilled water using 1.87 g glycine and 1.46 g NaCl. This buffer (81 ml) was further diluted to a total volume of 100 ml in 0.1 M HCl. To quantify chitosan, a dye solution was prepared. An aliquot of 150 mg Cibacron Brilliant Red 3B-A was dissolved in distilled water (100 ml). The glycine buffer was used to dilute 5 ml of the dye solution to a total volume of 100 ml. An aliquot of 300 μl of diluted vesicle suspensions (distilled water, 1:1, v/v) were mixed with 3 ml of the diluted Cibacron dye and surface-available chitosan was quantified using Spark M10 multimode plate reader (Tecan Trading AG, Männedorf, Switzerland) at 575 nm (Jøraholmen et al., 2015 (link)).

The total GSH was measured using a glutathione assay kit (Cayman Chemical), following the Tietze recycling assay method²⁶ and according to the manufacturer's instructions. Briefly, the cells were seeded in 96‐well plates (Nunc, 40 × 10³ cells/100 μL media/well) and incubated overnight in their normal media in a 37°C, 5% CO₂ humidified atmosphere. BSO (100 μM) was added to cells overnight where appropriate. The cells were incubated with 5‐ALA (0.2 mM T47D and MDA‐MB‐231, 0.5 mM MCF7, 1 mM T98G, and 2 mM U87) for 4 hours in the presence and absence of BSO, washed twice in PBS with calcium and magnesium, and finally left in complete medium. One hour following the addition of the medium, the cells were either treated with 0.5% MPA (for the glutathione assay) or 0.1% (v/v) Triton‐X 100 aqueous solution for protein quantification. The cells were then kept at −20°C until the GSH assay was performed. Prior to the glutathione assay, 2 μL of 4 M TEAM was added to each well treated with MPA to reinstate the physiological pH (7.4). Part of the supernatant (50 μL) from each well was moved to a new 96‐well plate and assayed for GSH content. The kinetic absorbance measurement was performed at 405 nm using the Tecan spark M10 plate reader. Blank values measured in wells with no cells were subtracted in all cases.

Differentiated cells in medium supplemented with a single protein or a combination of proteins were randomly assigned to at least 2 wells in each of the studied plates. In addition to blanks (medium only), undifferentiated cells, and differentiated cells without an added protein served as negative controls.
Quantitative analysis of the cellular insulin content in each well was carried out by a fluorescence microplate reader (Spark M10, Tecan GmbH, Grödig, Austria). Initially, the plate geometry edit was performed to verify the plate configuration using an empty plate. The following parameters were selected from the supplied Spark Control Magellan Software (Tecan, Austria) to detect fluorescence intensity. The high-energy xenon lamp was set up to flash 30 times. A wavelength of 485 nm was used for excitation, and a wavelength of 535 nm was used for emission in the bottom reading mode with sixteen multiple area reads/well. The mean fluorescent dye intensity of each well was calculated following blank reduction. The results were expressed as the numerical fluorescence intensity and as a colorimetric index.

A 96-well plate was coated with streptavidin,
o/n, at 37° (1 μg/mL in carbonate-bicarb buffer, pH 9.6).
Ten μg/mL of peptide in carbonate buffer was added to the plate
and incubated for 1 h at RT. Washing steps were performed with PBS
containing 0.05% Tween 20. The primary antibody (3F3 antibodies from
Theresa Kissel) and secondary antibody (goat anti-Human HRP, 1:5000,
Abcam, ab97225) were diluted in PBS containing 1% BSA and 0.05% Tween
20, and sequentially incubated for 1 h at 37 °C. Results were
analyzed with a Tecan Spark M10 plate reader.

Coacervation of K₇₂ and nucleotides ADP or ATP was always assessed with a commonly used turbidity assay, combined with microscopy. The absorbance at 600 nm was measured using a plate reader Spark M10 (Tecan), for samples containing: 25 mM HEPES pH 7.4, 20 μM K₇₂, 1 mM MgCl₂, and a varying concentration of ADP or ATP ranging from 1 to 10 mM. The samples were prepared on a 30-μL scale and placed in a 384-well plate (Nunc, flat bottom). Absorbance (Abs) was measured before and after 2 μL additions of NaCl 0.5 M, until it reached the value of the control lacking any nucleotide. Turbidity (%) was calculated as 100 (1 − 10^-Abs). Critical salt concentration was calculated using the last three values of absorbance measured to extrapolate the concentration needed for Abs = 0 (relative to the control).

The glycosyltransferase activity of the CslA–GlxA complex was quantitatively measured by using the UDP-Glo glycosyltransferase assay kit (Promega). This assay detects the content of free UDP, which is released during the glycosyltransferase reaction. After the separation of the insoluble glucans by centrifugation, 15 µL of the UDP-containing supernatant was mixed with 15 µL of freshly prepared UDP-Glo Detection Reagent, which also terminates further glucan synthesis. After 1 h of incubation at room temperature in the dark, luminescence was measured using a TECAN Spark M10 plate reader. All values were corrected using the enzyme blank controls, and the standard curve line of luminescence-UDP was used to convert measured luminescence to the UDP concentration. All measurements were performed in triplicate.