细胞抗氧化方法CAA步骤-康奈尔大学刘瑞海实验室实验方法.docVIP

Cellular Antioxidant Activity in HepG2 Cells A. Subculture of Cells 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 μg/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 Method: Cool the temperature to 4 °C in Beckman centrifuge. Keep all media, PBS, trypsin on ice during the whole procedure. Remove all growth media from 6-well plates or T75 flakes, and wash twice with 5 mL cold PBS. Remove all PBS from wells or flasks. 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. 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. Centrifuge 4 minutes at 650 rpm at 4 °C. 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.