Amphotericin b

The total count of yeast cells in the kombucha tea was determined using the spread plate technique on yeast malt (YM) agar with the addition of chloramphenicol. Determination of the total count of acetic acid bacteria was performed on yeast peptone mannitol (YPM) agar with amphotericin B (CAISSON, USA). The plates were incubated at 30 °C, for at least 3 days [14 ].

Three severely injured dogs (6–10 months) were presented at the outdoor clinic of the Faculty of Veterinary and Animal Science, PMAS-Arid Agriculture University, Rawalpindi, Pakistan. After proper examination and on advice of the registered hospital veterinarian, the dogs were euthanized for reasons unrelated to this study. Euthanasia followed the American Veterinary Medical Association (AVMA) guidelines for euthanasia of animals [44 ]. Briefly, a deep stage of anesthesia was achieved with intramuscular injection of xylazine HCL (1 mg/kg) and ketamine (5 mg/kg), in which cardiac arrest was induced by an intravenous injection of supersaturated solution of MgSO₄ (1 mL/kg). After euthanasia, adipose tissue (~ 3–5 g) from the inguinal subcutaneous (SC), omental (OM), and perirenal (PR) region and from the infrapatellar fat pad (IPFP) were isolated as presented in Fig. 1A-D, and kept in Dulbecco phosphate buffer saline (DPBS^−/−, i.e. without Ca⁺⁺ and Mg⁺⁺; Sigma-Aldrich, USA) with 5% penicillin (100 U/mL), streptomycin (100 μg/mL) and amphotericin-B (250 μg/mL, Caisson, USA) solution and transported to Stem Cell Physiology and Cytogenetic Laboratory at Faculty of Veterinary and Animal Sciences, PMAS-Arid Agriculture University, Rawalpindi, Pakistan. All experimental procedures were performed in strict accordance with the guidelines of the Institutional Animal Ethics Committee.

Isolated T cells were suspended at 10⁶ cells/mL in RPMI 1640 media with L‐glutamine (Caisson Labs, Smithfield, UT, USA) supplemented with 10% fetal bovine serum (FBS; Sigma‐Aldrich), 1% (v/v) of penicillin/streptomycin solution, 2.5 μg/mL amphotericin B (Caisson Labs), 25 mM HEPES (Caisson Labs), 100 ng/mL Interleukin (IL)‐2 (PeproTech, Cranbury, NJ), and 25 μL/mL ImmunoCult™ CD3/CD28 T cell activator (StemCell Technologies). Cells were seeded (10⁶ cells/well) in a non‐treated, flat‐bottom, 24‐well tissue culture plate (CellTreat, Pepperell, MA, USA) and incubated at 37°C with 5% CO₂ for 3 days. On day 3, cells were collected from the plate, spun down by centrifugation at 250 × g for 10 min, re‐suspended in culture media without activator (0.25 × 10⁶ cells/mL), and seeded in a new 24‐well plate (0.25 × 10⁶ cells/well). Culture media supplemented with 100 ng/mL of IL‐2 was replaced every 3 days. When the total cell count for the sample reached 10⁷, the cells were re‐suspended at a concentration of 10⁶ cells/mL and culture continued in a 250 mL suspension culture flask (CellTreat, Pepperell, MA, USA). Total numbers of viable cells were determined using an automated cell counter with Trypan Blue (Countess II, ThermoFisher, Waltham, MA) before each media exchange.

The human gastric cancer cell lines, AGS and NCI-N87 were purchased from Food Industry Research and Development Institute (Hsinchu, Taiwan). The human gastric cancer cell lines, HR and MKN45, and the duodenal cancer cell line AZ521 were provided by Prof. Chia-Jui Yen (Department of Medical Oncology, National Cheng Kung University Hospital, Tainan, Taiwan). The human leukemic monocyte lymphoma cell line U937 was a kind gift from Prof. Ming-Derg Lai (Department of Biochemistry and Molecular Biology, College of Medicine, National Cheng Kung University, Tainan, Taiwan). All the cell lines were maintained in RPMI-1640 medium (HyClone) supplemented with 10% fetal bovine serum (HyClone), 15 mM HEPES (Biological Industries), 2 mM L-glutamine (Caisson), and 1x antibiotic-antimycotic solution (1,000 units/L penicillin, 2.5 µg/L amphotericin B, and 1000 µg/L streptomycin) (Caisson) and were cultured at 37 °C in a humidified atmosphere containing 5% CO2 and 95% air.