Nt1310 Unit 9 Lab Report
3.4 Lift, Dissociate, and Pellet NPCs for Maintenance and/or Plating for Experimental Conditions
3.4.1 Aspirate medium, wash cells once with 1x PBS. Aspirate PBS and add 500 L of 1x Cell Detachment Solution into 1 well of confluent NPCs. Incubate for 10 min at 37 °C.
3.4.2 Add 500 L of room temperature (RT) PBS and wash the well using a P1000 to ensure removal of cells. Collect the cell + PBS solution into a 15 mL conical tube. Wash the plate again with 1 mL of PBS and add liquid to the tube.
3.4.3 Spin the cells down at 300 x g for 5 min to pellet.
3.4.4 Remove supernatant from the cell pellet and re-suspend the cells in 1-5 mL of pre-warmed DMEM/F12 media. Dilute cells to a density of 1 to 4 million cells/mL of media. Quantify cells using a hemocytometer.
3.4.5 …show more content…
3.4.6 Adjust cell suspension volume with media so that between 15 to 100 L of cells are used for each well/dish. This small plating volume ensures that there is even cell distribution and that growth factors, drugs, or substrates in the medium are not diluted.
3.4.7 Incubate cells at 37 °C. Change media every 48 h for NPC maintenance or see the specific details for individual
3.4.1 Aspirate medium, wash cells once with 1x PBS. Aspirate PBS and add 500 L of 1x Cell Detachment Solution into 1 well of confluent NPCs. Incubate for 10 min at 37 °C.
3.4.2 Add 500 L of room temperature (RT) PBS and wash the well using a P1000 to ensure removal of cells. Collect the cell + PBS solution into a 15 mL conical tube. Wash the plate again with 1 mL of PBS and add liquid to the tube.
3.4.3 Spin the cells down at 300 x g for 5 min to pellet.
3.4.4 Remove supernatant from the cell pellet and re-suspend the cells in 1-5 mL of pre-warmed DMEM/F12 media. Dilute cells to a density of 1 to 4 million cells/mL of media. Quantify cells using a hemocytometer.
3.4.5 …show more content…
3.4.6 Adjust cell suspension volume with media so that between 15 to 100 L of cells are used for each well/dish. This small plating volume ensures that there is even cell distribution and that growth factors, drugs, or substrates in the medium are not diluted.
3.4.7 Incubate cells at 37 °C. Change media every 48 h for NPC maintenance or see the specific details for individual