Materials. Minimum essential medium alpha modification (α-MEM) with L-glutamine (2.0 mM), penicillin-streptomycin, trypsin ethylene diamine tretaacetic acid (EDTA), 6- thioguanine (6-TG) were purchased from Sigma-Aldrich. Fetal bovine serum (FBS) was purchased from Hyclone (Logan, UT). Dimethylsulfoxide (DMSO) and potassium chromate (K2CrO4) were purchased from Fisher Scientific. Reagents such as crystal violet, 70% ethanol were used as is. Cells were stored in liquid nitrogen until ready for use. Particle counter was purchased from Beckman Coulter (Miami, FL). 60 x 15 mm and 100 x 15 mm cell culture dishes was purchased from VWR. Air incubator of 5% CO2 was purchased from Bacharach Co. (Pittsburgh, PA).
Preparation of medium alpha modification …show more content…
The mixture was prepared by mixing 100 mL of PBS and filled to 1 L with nanopurewater.
Preparation of trypsin ethylene diamine tetraacetic acid (EDTA). The 1:10 dilution is prepared by adding trysin EDTA (1 mL x 10) and prepared 10% PBS to be stored at -20 …show more content…
The cell culture of AA8 strain of Chinese hamster ovary (CHO, American Type Culture Collection, Manassas, VA) cells are obtained by removing the cryovial cell from liquid nitrogen and thawed in a water bath (37 oC) and sterilized with 70% ethanol. The cells are centrifuged with modified α-MEM (50 mL) and resuspended three times. The cells are incubated for 72 h at 37 oC in order to be placed in a culture hood and be harvested. Using the Coulter counter (Beckman Coulter, Miami, FL) to obtain a cell count while being incubated for 72 h at 37 oC then the volume of suspension of α-MEM can be determined so that each dish will hold 5 mL. 126 or more dishes will be seeded and incubated for 24 h at 37 oC. After incubation the dishes are placed in the culture hood to be treated with either EOC from the PM2.5, a negative control, or a positive control. Each set of three dishes will be treated with EOC volumes of (0, 10, 25, 50, and 100 μL), 50 μL of negative control, and 30 μM of positive control. Once cells are treated they are incubated for 24 h and harvested to obtain a new cell