User:David Dreher/Notebook/Chromatin controlled cell pattern/2013/02/15

02/15/13

• HEK293-luc cell culturing, trial 2

HEK293-luc #4 cell culturing, trial 2

• Tried plating at the optimal density (1600 cells/well) in a 6-well plate; cells did not grow very well
• Retry 6-well plates and also try 96-well plate
• Made three new plates of HEK293-luc #4 cells
• Started with ~1x10⁶ harvested cells (confluent T25 flask)

• Made the following dilutions:
  • (1) 1:25 dilution of harvested cells (1mL cells + 24 mL medium) = 40,000 cells/mL 10% FBS DMEM
  • (2) 1:25 dilution of previous tube (1mL 1:25 cells + 24 mL medium) = 1:625 dilution = 1,600 cells/mL 10% FBS DMEM
  • (3) 1:100 dilution of harvested cells (100 μL cells + 9.9 mL medium)
    --> 1:100 dilution of these (100 μL cells + 9.9 mL 20% FBS DMEM)
    --> 1:4 dilution of these (5 mL cells + 15 mL 20% FBS DMEM)
    = 25 cells/mL = 5 cells/200 μL (20 mL total)

Plates:

1. 6-well plate: 2 mL of 1:25 dln., ~80,000 cells/ well
2. 6-well plate: 2 mL of 1:625 dln., ~3,200 cells/ well
3. 96-well plate: 200 uL of 1:40,000 dln., ~5 cells/ well

Notes:

• Was careful to gently mix stock tube by inverting after every column was filled to ensure that cells stayed suspended in the stock tube.
• Used 20% FBS for 96-well plate to encourage growth of isolated/ low-concentration cells.