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…
Plate the required number of cells specific for either NPC maintenance (Section 3.3) or the individual assay being conducted (see subsequent sections for more details).
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
assays.
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…
Plate the required number of cells specific for either NPC maintenance (Section 3.3) or the individual assay being conducted (see subsequent sections for more details).
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
assays.