Spark m10 microplate reader

Chemokine-mediated recruitment of MiniG_αi or βarrestin2 to CCR7 was investigated by split luciferase assays [52 (link)], which rely on the complementation of two NanoLuciferase subunits upon close proximity of the two proteins of interest. In brief, transiently transfected HeLa cells were washed with PBS supplemented with 5 mM glucose (PBS-G), detached using PBS-G supplemented with 0.5 mM EDTA and collected with DMEM. Cells were then washed and resuspended in PBS-G and loaded with 5 µM of the luciferase substrate CoelenthrazineH (Biosynth, Staad, Switzerland). After distribution into a 96-well half-area plate, baseline luminescence was measured for 10 min (384–440 nm, 500 ms integration time) before stimulation with the indicated concentrations of chemokine. Luminescence was further recorded over a period of 30 min on a Spark M10 microplate reader (Tecan, Männedorf, Switzerland).
For characterizing the effect of different glycosaminoglycans on chemokine signaling, cells were simultaneously stimulated with different concentrations of chondroitin sulfate A (Roth, Arlesheim, Switzerland), heparin (Roth), N-acetyl neuraminic acid (Roth) or BSA (Roth).

Uncleared and cleared adipose tissue was immersed in 100 µl of CUBIC2 reagent in a 96-well clear-bottomed microplate (Greiner). Tissue absorbance measurements were taken every 2 nm between 400 nm and 1000 nm with the Spark M10 Microplate Reader (Tecan). Blank measurements were taken from an equal volume of CUBIC2 reagent in the same 96 well plates. To calculate tissue transmittance (T) from absorbance (A) we used the equation T = 10^(−A) and calculated the mean of three individual measurements of the same tissue. Results are presented as percentage of normalized transmittance to blank, which is set at 100%.
Measurement of tissue biopsy area before, during and after dehydration and rehydration steps and optical clearing was made with ImageJ. The length of the scale bar in the images (1 cm) was used to set the reference scale for measurements. The tissue perimeter was drawn using the area selection tool and the area was calculated in cm² using the measure function.

The effect of in vitro acetylation on bacterial protein synthesis was investigated in the Expressway cell-free E. coli expression system (Invitrogen). The green fluorescent protein (GFP) gene was amplified from the pCmGFP plasmid (47 (link)) and inserted into pEXP5-CT TOPO (Thermo Fisher Scientific), and the resulting plasmid was used as a template for in vitro transcription and translation (IVTT). Reactions were performed according to the manufacturer’s protocol, in 50 μL of reaction mixture, in the wells of a black polystyrene 96-half-well microplate (Greiner). Feed buffer was added after 30 min of incubation, to ensure optimal protein synthesis. Reactions were initiated by adding 1 μg of plasmid DNA. The plate was incubated at 30°C and fluorescence was measured every 20 min (λ_ex = 475 nm; λ_em = 520 nm) with a Spark M10 microplate reader (Tecan). Fluorescence from GFP synthesis was recorded at 20 min after addition of feed buffer using GraphPad Prism 6 software. Gentamicin was used as positive control at 10 μM.