Sodium bicarbonate

Pure cultures of Str. pyogenes, S. aureus, S. epidermidis, P. aeruginosa, and Ent. Faecium were used for the study of antibacterial activity using the method adapted from Koller M. et al. [31 (link)].
Before starting, standard solutions containing 2 × 10⁶ CFU/mL each were prepared in RPMI-1640 medium (Thermo Fisher Scientific, USA) with the addition of 0.3 g/L L-glutamine and 2 g/L sodium bicarbonate (Thermo Fisher Scientific, USA), and samples were placed in wells of a 6-well plate. Fifty microliters of the bacterial suspension was applied as a drop to each test sample and incubated in a humid thermostat at 37 °C (Figure 1). The next day, a drop of bacterial suspension from the sample was transferred to a Petri dish with appropriate culture medium and cultured further for 24 h to evaluate bacterial growth. Bacterial colonies were stained with Hoechst 33342 fluorescent dye (Thermo Fisher Scientific, USA) and were counted using an Axioskop 40FL microscope (Carl Zeiss, Jena, Germany). Measurements in each experimental group were performed in three repetitions.

Untreated jute fibers were provided by Toptrans Bangladesh Ltd. (Dhaka, Bangladesh). Fibers were cut to 3–5 cm in length and further grinded by an IKA MF 10 basic grinder at 1000 rpm (IKA Works Inc., Wilmington, NC, USA). Analytical-grade nitric acid (ACS reagent 60%) and sodium nitrite (ACS reagent ≥ 97%) were purchased from Sigma-Aldrich (Allentown, PA, USA); sodium bicarbonate was purchased from Fischer Scientific (Fairlawn, NJ, United States). Processed polygen liquid latex (NRL) nonvulcanized with 60% concentration was obtained from UK suppliers (ReAgent, Runcorn, UK).

GM-CSF and CXCL8 were obtained from R&D Systems. Histopaque was purchased from Sigma-Aldrich. EDTA, Sodium Bicarbonate, 10X MEM, RPMI and HEPES were all obtained from Fischer Scientific. PI3Kδ selective inhibitor (PIK-294) was purchased from Symansis. Wortmannin, PI3Kγ selective inhibitor (AS-605240) and PI3Kα selective inhibitor (PIK-75) were from Calbiochem (Merck). Phospho-Akt and Akt were purchased from Cell Signalling Technology. Collagen I was purchased from Nutacon.

Human embryonic kidney (HEK) 293FT cells were cultured in complete Dulbecco’s modified Eagle’s medium (DMEM) containing 1% DMEM powder (Gibco, #31600091), 0.35% (w/v) d-glucose (Sigma-Aldrich, #50-99-7), 0.37% (w/v) sodium bicarbonate (Thermo Fisher Scientific, #S233-500), 10% heat-inactivated fetal bovine serum (Gibco, #16140071), 4 mM l-glutamine (Gibco, #25030081), and penicillin (100 U ml⁻¹) and streptomycin (100 μg ml⁻¹) (Gibco, #15140122) in tissue culture–treated 10-cm dishes (Corning, #500001672) at 37°C in 5% CO₂. To passage, medium was aspirated, and cells were washed in phosphate-buffered saline (PBS), incubated in trypsin-EDTA (Gibco, #25300054; 37°C, 5 min), detached by tapping the dish, and resuspended in fresh medium and plated. This cell line tested negative for Mycoplasma using the MycoAlert Mycoplasma Detection Kit (Lonza, #LT07-318).

Cryopreserved human primary hepatocytes (Lonza) were plated overnight on collagen-coated 12-well plates at 1 × 10⁵ cells per well in MM (Lonza). 24 h after plating, cells were serum-starved in DMEM base medium (Sigma) supplemented with 1 g/L glucose (Sigma), 3.7 g/L sodium bicarbonate (Sigma), and 4 mM L-glutamine (Corning) overnight, followed by 24 h incubation in 0.3 ml glucose-production medium: DMEM base with 2 mM glutamine, 3.7 g/L sodium bicarbonate, 15 mM HEPES (ThermoFisher), 20 mM lactate (Sigma), 2 mM pyruvate (Fisher) and 0.1 mM pCPT-cAMP (Sigma). After 24 h, 50 μL of medium was removed for glucose detection with Invitrogen glucose Colorimetric Detection kit (#EIAGLUC), according to manufacturer’s protocol, and read on a plate reader (Multiskan GO, Thermo-Scientific). Because hepatocytes were extensively washed prior to cell incubation in glucose-free media for this assay, the only potential source of glucose in the media is hepatic production. The prolonged culture of cells in low glucose media prior to the assay depletes hepatocytes of glycogen stores. The media used during this assay contains high concentrations of gluconeogenic substrates, primarily lactate, favoring gluconeogenesis^{67 (link),68 (link)}.