Spectrofluorometer

Ca²⁺ influx was measured by recording [Ca²⁺]_i with Fura-2-AM labelling (F1201; Invitrogen, Molecular Probes, Eugene, Oregon, USA) as described previously [17 (link)]. Briefly, WT and STIM1^−/− microglia grown on glass cover slips were loaded with cell-permeable Fura-2-AM, and trapped Fura-2 fluorescence was measured with a spectrofluorometer (Photon Technology International, Birmingham, NJ, USA). Cells were perfused with a solution containing 150 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 10 mM glucose, 10 mM HEPES (pH 7.4 adjusted with NaOH) containing either 1-2 mM CaCl₂ or 0.5 mM EGTA (to chelate any Ca²⁺ in the solution). The osmolality of all solutions was adjusted to 310 Osm with the major salt. The Fura-2 ratio was recorded using dual-excitation wavelengths at 340 and 380 nm, and emission wavelengths above 510 nm were monitored. Cells were treated with 25 μM cyclopiazonic acid (CPA; C1530, Sigma Aldrich, St. Louis, MO, USA), which inhibits the endoplasmic reticulum (ER) Ca²⁺-ATPase. Cells were treated with 100 μM uridine 5′-(trihydrogen diphosphate) sodium salt (UDP; U4125, Sigma Aldrich, St. Louis, MO, USA) or 50 μM adenosine 5′-triphosphate di(tris) salt hydrate (ATP; A9062, Sigma Aldrich, St. Louis, MO, USA).

Biochemical measures were taken 4 months after surgery. Cytosolic proteins were extracted in lysis buffer (1% Triton X-100, 0.32 M Sucrose, 10 mM Tris (pH 8.0), 5 mM EDTA, 2 mM DTT, 1 mM PMSF, 10 µg/mL Leupeptin, 10 µg/mL Pepstatin A, 10 µg/mL Aprotinin). Enzymatic reactions were produced in reaction buffer (50 mM Tris (pH 7.5), 5 mM MgCl₂, 1 mM EGTA, 0.1% CHAPS, 1 mM DTT) with 25 µg of proteins and fluorogenic substrate: Ac-DEVD-AMC (40 µM) for caspase-3, and Ac-VEID-AMC (40 µM) for caspase-6. Reactions were incubated at 37 °C for 180 min for caspase-3 and 90 min for caspase-6 and stopped with the addition of 0.4 M NaOH and 0.4 M glycine buffer. Fluorescence was quantified with a spectrofluorometer (Photon Technology International, Lawrenceville, NJ, USA) at excitation wavelength of 365 nm (caspase-3) or 325 nm (caspase-6) and emission wavelength of 465 nm (caspase-3) or 435 nm (caspase-6).

Determination of the dissociation constant for the complex formation between the different forms of the compound 1 and 5 was achieved by analyzing the change of the intrinsic autofluorescence of the flavin group of the reductase after complex formation, as previously used for other ligands of the reductase [6 (link),7 (link)]. The fluorescence intensity of the Cb₅R after the sequential addition of increasing amounts of compound to phosphate buffer saline buffer (PBS) in the presence of Cb₅R (0.25 µM) while stirring at pH 7.0 at 25 °C was monitored using a spectrofluorometer (Photon Technology International Inc. Quantamaster, Ford, U.K.) and fixed excitation and emission wavelength at 470 and 520 nm and using a 2 mL quartz cuvette. The obtained data after titration were adjusted to the following equation for the calculation of the K_d for the complex formation:
where F is the Cb₅R fluorescence change induced by the addition of the compounds to the cuvette, F₀ is the initial Cb₅R fluorescence and F_max is the fluorescence increment after adding the compounds, and K_d is the dissociation constant value.

Fluorescence based in vitro transcription assay was described (Datta et al., 2016 (link)). 400 ​nM Mtb. RNAPσ^Aholo was incubated in 2 μl transcription buffer (50 ​mM Tris-HCl (pH 8), 100 ​mM KCl, 10 ​mM MgCl₂, 1 ​mM DTT, 50 ​nM BSA and 5% glycerol) at 25° Celsius for 15 ​min to reactivate. 5 ​ng/μl of pUC19-sinP3 plasmid DNA along with 0, 10, 20, 40, 80 ​μg/ml of the phyto-chemical CU1, was further added and incubated at 37° Celsius for 20 ​min to form open promoter complex (RPo). RNA synthesis was initiated by the addition of NTP mix (final concentration 250 ​μM of ATP, GTP, CTP and UTP) and incubated for elongation at 37°Celsius for 30 ​min. The reaction was stopped by adding 5U of RNase-free DNase I (Thermo Scientific), followed by incubation for 1 ​h at 37° Celsius. RiboGreen dye (Invitrogen, Carlsbad, CA) diluted to 200 folds in TE buffer (10 ​mM Tris-HCl pH 8.5, 1 ​mM EDTA) was prepared. After DNase I digestion, the samples were incubated for 5 ​min at 25° Celsius with the prepared RiboGreen dye solution in TE buffer, so that the reaction mix get 10-fold diluted. Fluorescence intensities of the samples were monitored using a spectrofluorometer (Photon Technology International, HORIBA Scientific, Edison, NJ) at excitation and emission wavelengths of 500 and 525 ​nm, respectively (Mukhopadhyay et al., 2003 (link)).

Samples were prepared by placing 3 ml of PBS buffer in a cuvette, 75 µl of the liposomes were added to the same cuvette and were mixed gently. Calcein release from the liposomes was triggered using a low-frequency ultrasonic (LFUS) probe at 20 kHz (VCX750, Sonics & Materials Inc., Newtown, CT) using a power density of 10 mW/cm² in a pulsed mode (20 s on and 10 s off). Online monitoring of calcein fluorescence following its release from the liposomes was conducted at room temperature at excitation wavelengths of 495 nm and emission wavelength of 515 nm (slit width of 5 nm) using a spectrofluorometer (Photon Technology International, Edison NJ, USA). The initial fluorescence intensity, I_o, was measured for 70 s before sonication. Once a fluorescence plateau was reached, 50 μl of Triton X-100 were added to the sample to lyse the liposomes and achieve 100% calcein release. The following equation was used to calculate the cumulative fraction release (CFR) of calcein: $$CFR=\frac{I_t-I_o}{I_{\infty }-I_o}$$ where I₀ stands for calcein fluorescent intensity before applying ultrasound, I_t, stands for calcein fluorescent intensity at time, t, and I_∞ is the highest recorded calcein fluorescence intensity.