Alamar blue

Malic acid, Diosgenin, Pluronic F68, 1, 12-Dodecanedioic acid (DDA), Pluronic 127 were purchased from Sigma Aldrich. Acetone was obtained from Sisco Research Laboratories Pvt. Ltd. (Mumbai, India). Perchloric acid was purchased from Central Drug House (CDH) (Mumbai, India). For animal cell culture studies, Phosphate buffered saline, Alamar Blue and DAPI or 4′,6-diamidino-2-phenylindole, sodium bicarbonate were procured from HiMedia (Mumbai, India). Dulbecco’s modified Eagle’s medium (DMEM), Fetal Bovine Serum (FBS), Trypsin-EDTA (0.25%) were purchased from Gibco Life Technologies (Carlsbad, CA, USA). MilliQ water was used in the preparation of solutions during the study. A549, human adenocarcinoma cell lines were purchased from NCCS, Pune, India.

Madhugrit (batch #1MDT-210081) was sourced from Divya Pharmacy, India. The standards for HPLC analysis namely gallic acid, magnoflorine, piperine, rutin, ellagic acid, coumarin, cinnamic acid, and palmatine were obtained from Sigma-Aldrich, USA; protocatechuic acid, corilagin from Natural remedies, India, and methyl gallate from TCI chemicals, India. Reagents namely RPMI 1640 (no glucose), DMEM (low glucose), DPBS, antibiotic-antimycotic solution, starch, Malondialdehyde (MDA), Trichloroacetic acid (TCA), LPS, and 2′,7′-Dichlorofluorescin diacetate (H₂DCFDA) were procured from Sigma-Aldrich, USA. Heat-inactivated FBS, TPVG, Bovine serum albumin (BSA), Nile red, and Alamar blue were obtained from HiMedia, India. Horse serum was bought from Gibco, USA. Chemicals, 2-thiobarbituric acid (TBA), D-glucose, Metformin, and dexamethasone were obtained from TCI chemicals, India. Phorbol 12-myristate 13-acetate (PMA) was purchased from Alfa Aesar, UK. Mitomycin C was obtained from Roche, Germany. Pierce BCA protein assay kit and sodium azide were obtained from Thermo Fisher Scientific, USA. Human TNF-α and IL-6 ELISA kits were obtained from BD Biosciences, USA. QUANTI-Blue reagent was purchased from InvivoGen, USA.

Cell viability was measured by Alamar blue assay [27] (link). Briefly, Alamar blue (Resazurin sodium salt from Himedia) was dissolved in phosphate buffered saline pH 7.4 to make a stock of 5 mg/mL and a final working concentration of 0.1 mg/mL in cell culture medium. Resazurin is a redox indicator, which measures the reducing environment of the cell by reducing to a highly fluorescent resorufin. Astaxanthin at different concentrations (5, 10, 20, 50, 100, 200 and 400 µM) was added to the cells and after 24 h, Alamar blue dye was added and the plates were incubated at 37°C for 4 h. The color change was monitored colorimetrically at 595 nm and 570 nm to evaluate oxidized versus reduced forms respectively of the reagent by using multi-mode plate reader.

We followed the protocol of Collins and Franzblau (1997 (link)) with slight modifications. Briefly, freshly grown mycobacterial cultures (~ 1 OD₆₀₀ in 10 ml rich medium) were washed once with fresh sterile 10 ml rich medium and subcultured to 0.05 OD₆₀₀ in 10 ml fresh rich medium. When OD₆₀₀ reached 0.4–0.6, the cells were washed once with the fresh rich medium at 4,000 rpm and RT for 15 min and then used for estimation of MIC. Approximately 3.75 to 4 × 10⁴ colony-forming units (CFUs) were used for the assays. Two-fold dilutions of different drug concentrations (INH:0.02 to 23.3 μM; RIF:0.75–768 nM; EMB:0.15–156.63 μM) were added in triplicates to different wells with appropriate controls (positive-culture only; negative-medium only, and drug only). The final volume in each well was made up to 200 μl with the rich medium and gently mixed 3–4 times with a 200-μl pipette. After 5 days of incubation at 37°C (without shaking), 22 μl of 10× alamarBlue (HiMedia) was added, and the contents were mixed again before incubation (without shaking) for additional 2 days. Color change from blue (metabolically inactive/dead) to pink (metabolically active/growing) was photographed and recorded. To prevent loss of medium due to evaporation in experimental wells, all the peripheral wells were filled with 200 μl of sterile rich medium.

M.smegmatis mc²155, initially obtained from ATCC, was maintained in our laboratory as glycerol stocks. M.tb H₃₇Rv (gift from Prof R. K. Bhatnagar, Jawaharlal Nehru University, New Delhi, India), was cultured in BSL-3 facility. Growth media (Middlebrook 7H9) and OADC supplement were obtained from BD, USA. glycerol, Tween 80, acetic acid, acarbose and cyclosporine-A were procured from Sigma-Aldrich, India. GaNP (purity > 99.9%) was obtained from Nanoshel, India. All other reagents such as Alamar Blue, Crystal Violet, Isoniazid, and Ethambutol were of analytical grade and obtained from Himedia, India.