Streptomycin sulphate

Dulbecco’s Modified Eagles medium (DMEM), rhodamine-123, fetal bovine serum (FBS), tris-EDTA buffer, propidium iodide (PI), ethidium bromide (EtBr), annexin-V, 4′,6′-diamino-2-phenylindole, fluorescein isothiocyanate (FITC), amphotericin B, phosphate buffered saline (PBS), 2′,7′-dichlorofluorescein diacetate (H₂DCF-DA), penicillin G sodium, streptomycin sulphate, and lactophenol were purchased from HiMedia Laboratories, Mumbai, India. The other organic solvents and fine chemicals were of analytical grade and were purchased from Merck and SD Fine Chemicals, Mumbai, India.

Surface Sterilization for isolation of endophytic fungi was done following previously established procedures [24 ]. Washed plant parts were treated by the following immersion sequence: 70% ethanol for 2 mins followed by 1% sodium hypochlorite (NaOCl) solution for 3 mins. Thereupon, samples were rinsed in double distilled, sterilized water for a couple of minutes. Then samples were dried on a blotting sheet. Imprints of dried and sterilized samples were taken on media plates; finally samples were chopped into 8 mm diameter segments and placed (3–4 segments on each plate) onto petri dishes (Tarsons, Kolkata) containing malt agar as medium. Malt extract-Agar medium {Malt extract (Himedia, Mumbai)15g, Agar powder (Himedia, Mumbai)15 g dissolved in 1000ml distilled water} amended with antibiotics chloramphenicol (Himedia, Mumbai) @ 0.2g/l and streptomycin sulphate (Himedia, Mumbai) @ 0.1 g/l of media at 7.4–7.8 pH was used as an medium for isolation, purification and maintenance of endophytic fungi. The plates were incubated at 24 ± 2°C for upto three weeks. The plant segments were observed once a day for the growth of the endophytic fungi. Hyphal tips of the endophytic fungi growing from the plant segments were isolated from isolation plate and maintained on fresh malt agar plates and coded with a unique number until identified.

The positively sorted CD14 positive cells were re-suspended in RPMI 1640 medium (Himedia, Mumbai; India) supplemented with 10% fetal bovine serum (FBS) (Biological Industries, Beit-Haemek Israel) and 1X antibiotic-antimycotic solution containing streptomycin sulphate, penicillin and amphotericin-B (Himedia, Mumbai; India), and plated at a density of 4 × 10
⁶ cells per well in 6 well low adherence plates (Corning, Tewksbury; USA). The cells were periodically harvested by gentle scrapping and passed through a magnetic column. Cells with and without bound microbeads (obtained in the flowthrough) were counted using a hemocytometer (Rohem Industries Pvt Ltd, India).