The human breast adenocarcinoma (MCF-7) and human cervical adenocarcinoma (HeLa) cell lines were maintained in MEM supplemented with 10% FBS, 1% antibiotics (100 U/ml penicillin, 100 [micro]g/ml streptomycin), and 250 [micro]g/ml fungizone
. The cells were grown statically at 37 [degrees]C in a humidified incubator set at 5 % C[O.sub.2].
Retinal explants were obtained from eye dissection in sterile CMF solution and placed in the wells with the RGC nerve fiber layer contacting the laminule, in 0.5 mL Neurobasal-A medium (Invitrogen), containing 0.4% methylcellulose (Sigma), 1% penicillin/streptomycin, 0.2% fungizone
, 0.5% L-glutamine, and 2% B-27 supplement for retina (all from Invitrogen).
At the end of the surgery, vancomycin (1 mg/0.1 ml), ceftazidime (2 mg/0.1 ml), and fungizone
(0.00549 mg/0.1 ml) were administered intravitreally and gentamicin (20 mg/0.5 ml) subconjunctivally.
In Group C no intervention was done for first 4 days, on 5th day rabbits were given acute nephrotoxic single dose of amphotericin B deoxycholate6 (inj Fungizone
, Bristol Myers Squibb, Turkey), 4mg/kg as slow IV infusion diluted in 5% dextrose water and sacrificed on 6th day.
Aliquots of frozen primary HEECs and HUVECs were thawed and grown to confluence in basal medium (BM), and a phenol red-free 1:1 v/v Dulbecco's Modified Eagle Medium/Ham's F-12 (Gibco, Grand Island, NY) mixture, containing 100 U/mL penicillin, 100 [micro]g/mL streptomycin, and 0.25 [micro]g/mL Fungizone
complex (Gibco) supplemented with 10% charcoal-stripped calf serum (Gibco).
Cells were cultured under standard conditions in [alpha]-MEM supplemented with 10% FBS, 0.25% fungizone
(Sigma-Aldrich, USA) and 1% gentamicin (Sigma-Aldrich).
A 450 [micro]L aliquot comprised of 400 [micro]L of Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, 100 U/mL of penicillin and streptomycin, and 1 U/mL of fungizone
and gentamycin was added to each tube, and the sample was homogenized using a micropestle.
Each well was filled with 350 [micro]L phenol red-free medium 199 (21157-029, Invitrogen Life Technologies, Saint Aubin, France) supplemented with 50 [micro]g/mL gentamicin (G1272, Sigma-Aldrich, Saint-Quentin, France) and 2.5 [micro]g/mL Fungizone
), a polyene antifungal, is an amphipathic fermentation product of the Gram-positive bacterium, first isolated from Streptomyces nodosus in 1955.
The biopsies were isolated using an 8.5 mm trephine drill (Smith & Nephew, Hoofddorp, NL) while cooling with sterile phosphate-buffered saline (PBS; Sigma, Zwijndrecht, NL) supplemented with 200 U/ml penicillin (Lonza, Westburg, Leusden, NL), 200 [micro]g/ml streptomycin (Lonza, Westburg, Leusden, NL) and 5 [micro]g/ml fungizone
(LifeTechnologies, Bleiswijk, NL).
Tissue explants were cut in approximately 4 pieces of 1 mm and washed 3-5 times with sterilized 1x PBS supplemented with 10% penicillin, streptomycin, and fungizone
Late passage cells, harvested after at least 100 passages, were seeded in 12-, 24-, or 96-well plates at densities of 20,000, 10,000, or 2000 cells/well, respectively, and cultured in DMEM (pH 7.6) supplemented with 5% fetal bovine serum, 2.5 mg/mL Fungizone
, and 100 U/L gentamicin in 5% C[O.sub.2]/95% air at 37[degrees]C.