细胞抗氧化方法CAA步骤-康奈尔大学刘瑞海试验室试验方法

Cellular Antioxidant Activity in HepG2 CellsCellular Antioxidant Activity in HepG2 Cells A.A.Subculture of CellsSubculture of Cells Materials:Materials: 1. Cell culture medium:HepG2----CM; MCP-7----DMEM 2. Cell wash medium: DMEM （2.5% FBS）with 10 mM Hepes, 50 unites/mL penicillin, 50 μg/mL streptomycin, and 100 μ g/mL gentamicin. 3. Trypsin-EDTA: 1X, 0.05%, GIBCO, Invitrogen Corporation, Lot:435993 4. Trypan blue Stain: 0.4%, Lot:347940, GIBCO, Invitrogen Corporation, Grand Island, NY, USA. 5. PBS 6. Beckman centrifuge GS-6R :: 1.Cool the temperature to 4 °C in Beckman centrifuge. 2.Keep all media, PBS, trypsin on ice during the whole procedure. 3.Remove all growth media from 6-well plates or T75 flakes, and wash twice with 5 mL cold PBS. 4.Remove all PBS from wells or flasks. 5.Add trypsin/EDTA to wells or flasks (0.6 mL/well for 6 well plates, and 2.5 mL per T75 flask). Trypsinize quickly –around one minute (1-- 1.5 min) at room temperature, or monitor cell starting shrink under microscope. 6.Dislodge cells: 1）6 well plate: using P1000 pipetman to dislodge cells by up-down blowing. It is easier to dislodge cells by pooling the trypsin solution from two wells (1mL per well ) and rinse once with the trypsin solution from another two wells. The purpose of up-down blowing is to obtain single cells. 2) T75 flask: Gently clapping the flask on side with your hands to dislodge cells and then using 5- mL pipet to dislodge cells by up-down blowing. It is easier to dislodge cells by pooling the trypsin solution from two flask (around 5 mL). Rinse once with washing medium. 3) Removing the cells into a 50 mL centrifuge tube containing around 20 mL of cold washing media ( 2.5% FBS/DMEM) 4) Wash the wells or flask with 2.5% FBS/DMEM (cold) and to centrifuge tube to a total volume of 35-40 mL. Mix well by up-down 3 times. 7.Centrifuge 4 minutes at 650 rpm at 4 °C. 8.Discard the supernatant and mix the cells by tapping the tube with your finger. When cells are homogenous, add 40 mL 2.5% FBS/DMEM and mix well by up-down. Centrifuge 4 min at 650 rpm at 4 °C. 9.Discard supernatant and mix cell pellet by tapping tube with finger. The cells are resuspended in 5 mL CM (FBS/DMEM ) (4 °C) and enumerated. ( For 2x6-well plates or 2x T 75 flasks, resupend the cells in 5 mL, and keep cell counts 20 per field). Keep suspension on ice Keep suspension on ice. 10. Add 50 μL of cell solution into 450 μL trypan blue dye. Calculate the cell number by counting at least 6 fields and averaging the counting. Cell count: 50 μL of cell solution + 450 μL of trypan blue Calculation: Cell # x 10 x 10,000 = cells/mL Total cell number: Cells/mL x total volume of cell solution 11. Calculate the amount of cells you need. Divide the number of cells you need by the number of cells you have = number of mL of cell suspension to use. ( It is easier to with 1.0 x 106 cells per mL). 12. Plate cells and culture at 37 °C in 5% CO2. 13. After seeding 0.5 h, very gently rock the plate/flask