User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/03/19

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Summary

To prepare for Co-IP, stimulation for ELISA, VASP and to keep the line alive cells were counted and split. Also cells were plated out for Anouk and cells were kept for Wolter to split for his own projects.

Materials & Methods

Materials

  • 500 mL warm DMEM S+
  • 450 mL warm DMEM
  • 10 mL PenStrep
  • 3 mL Fungizone
  • 50 mL FBS (HI)
  • 100 mL Warm PBS
  • 4x 15 mL tubes
  • 8x 50 mL tubes
  • 2x 24-wells plates
  • 4x Ø 10cm petridishes
  • 2x 6-wells plates
  • 16x Ø 15cm petridishes
  • 8 mL Trypin (5x diluted)

Method

Preparation of Medium

DMEM S+

  • Remove 50 mL of DMEM out of 500 mL
  • Add 50 mL FBS
  • 3 mL Fungizone
  • 10 mL PenStrep

Collecting cells

  • Warm solution required in water (PBS, DMEM S+)
  • Switch on laminar flow, clean it and spray everything required (except for cells) with EtOH (70%) prior to putting into the laminar flow
  • Switch the vacuum pump on
  • Check cells under microscope
  • Remove medium with vacuum pump
  • Rinse cells twice with 5 mL PBS and remove buffer with vacuum pump
  • Defrost trypsin
  • Add 1 mL trypsin
  • Incubate 37 °C, 5 min.
    • Prepare Greiner tube with 26 mL DMEM S+
    • Prepare plates with Name, Donor and Passage
  • After incubation add 5 - 10 mL DMEM S+ to cells (from prepared 26 mL)
    • Rinse the dish with the medium in order to get all cells removed from plate and in the liquid
  • Remove cell suspension and add to the remaining medium in the prepared Greiner tube (26 mL + 4×1 mL trypsin = 30 mL total volume)

Cell counting

  • Count cells using Bürker-Türk counting chamber (See image 120220101)
    • Put ~10 μL in each chamber and count the cells present in 4 large squares, selected randomly from both chambers.
  • The average of cells in the 4 blocks has to be multiplied by 104 (amount of cells per mL) and then by the volume in which the cells are resuspended (30 mL).
  • Clean the counting chamber with 70% EtOH
  • Correct the amount of cells by diluting with DMEM S+


  • D9
    • 1x 24-wells (25.000 cells/well (500 μL/well), 625000 cells/12.5 mL) --> Stimulation
    • 2x Ø 10cm (750.000 cells/plate (10 mL/plate), 1500000 cells/20 mL) --> Co-IP/Split cells
    • 1x 6-wells (200.000 cells/well (2 mL/well), 1400000 cells/14 mL) --> VASP
    • 8x Ø 15cm (1×106 cells/plate (15mL/plate), 3x 3×106 cells/45 mL) --> Anouk
  • D12
    • 1x 24-wells (25.000 cells/well (500 μL/well), 625.000 cells/12.5 mL) --> Stimulation
    • 2x Ø 10cm (750.000 cells/plate (10 mL/plate), 1.500.000 cells/20 mL) --> Co-IP/Split cells
    • 1x 6-wells (200.000 cells/well (2 mL/well), 1.400.000 cells/14 mL) --> VASP
    • 8x Ø 15cm (≤2×106 cells/plate (15 mL/plate), 3x ≤6×106 cells/45 mL) --> Anouk

Results

  • Cell counting (# of cells)
D12 Count #1 Count #2 Count #3 Count #4 Average Cells / mL
68 63 44 59 58,5 585000
D9 Count #1 Count #2 Count #3 Count #4 Average Cells / mL
50 65 61 73 62,25 622500


  • Preparing cell dilutions
D12 ' 24-wells 10 cm 6-wells 15 cm
Cells 585000 625000 1500000 1400000 6000000
mL Cells 1 1,07 2,56 2,39 10,26
mL S+ 11,43 17,44 11,61 34,74
SUM 1,00 12,50 20,00 14,00 45,00
D9 ' 24-wells 10 cm 6-wells 15 cm
Cells 622500 625000 1500000 1400000 3000000
mL Cells 1 1,00 2,41 2,25 4,82
mL S+ 11,50 17,59 11,75 40,18
SUM 1,00 12,50 20,00 14,00 45,00
  • Because the total volume was 30 mL, 3x 10.26 mL was not possible, instead ~7 mL was used.

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