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

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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.



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