We minced splenic tissue and passed it through a cell strainer in the presence of phosphate-buffered saline (PBS) supplemented with penicillin/streptomycin and fungizone
. We centrifuged the suspension at 4,000 x g for 30 min and stored the supernatant at 4[degrees]C.
Around 10 cm long hUCWJ was collected from the placental end in 50 ml tube containing 32 ml of Hank's Balance Salt Solution (HBSS) supplemented with 300 U/ml penicillin, 300 [micro]g/ml streptomycin, 150 [micro]g/ml gentamicin and 1 [micro]g/ml fungizone
. Under a laminar flow hood, the UC was transferred to Petri dishes which contain PBS.
The non-aggressive human mammary epithelial breast cancer cell line MCF-7 was purchased from VACSERA (Cairo, Egypt) and cultured in RPMI (Roswell Park Memorial Institute; Buffalo, New York, USA) medium supplemented with 100 ug/mL penicillin-streptomycin, 2.5 ug/mL fungizone
, 10% heat-activated foetal calf serum, and 2 mM glutamine.
Mouse melanocytes (B16F10) were cultured in complete Minimum Essential Eagle's Medium (MEM), containing 10% FBS, 1.5 g/L NaHC[O.sub.3], 2 mM L-glutamate, 10 mg/ml streptomycin, and 0.25 mg/ml fungizone
Samples were transferred to the lab in a transfer medium comprising DMEM/F12, 100 [micro]g/ml penicillin, 100 IU/ml streptomycin and 0.25 [micro]g/ml fungizone
(Invitrogen, Carlsbad, CA).
DMEM-high glucose, FBS-heat inactivated, Penicillin/Streptomycin, and Fungizone
were from Biowest (USA).
Retinal pellets were resuspended in DMEM F12 containing B27-supplement (1:50), 0.25 [micro]g/mL fungizone
, 100 [micro]g/mL penicillin/streptomycin, and methylcellulose (1 : 250).
The suspension was then centrifuged and washed three times (1000 g; 10, 10, and 5 min) in PBS with 1% antibiotic-antimycotic solution (AAS) containing penicillin, streptomycin, and fungizone
(Thermo Fisher Scientific, USA).
The human breast cancer cell line MCF-7 and T47D cells were purchased from VACSERA (Cairo, Egypt); growth conditions of these cells are full RMPl medium supplemented with 5% (v/v) foetal calf serum, 10 [micro]g/ml penicillin-streptomycin, and 2.5 [micro]g/ml fungizone
. Hereafter referred to as RPMl + 5%.
The olfactory mucosa was then collected from both sides using a sterile needle, and fragments were pooled into a sterile 2 mL tube filled with 37[degrees]C DMEM/Ham's F12 culture medium supplemented with 10% fetal bovine serum, 200 units/mL of penicillin, 200 [micro]g/mL of streptomycin (all products from Life Technologies, France), 1.25 [micro]g/mL amphotericin B (Fungizone
, Sigma-Aldrich, France), and 12.5 [micro]g/mL of Plasmocin treatment (InvivoGen, France).