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 Counting hTERT cells
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Summary
- 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
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
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)
- 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
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- 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
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Results
- Cell counting (# of cells)
| D12
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Count #1
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Count #2
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Count #3
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Count #4
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Average
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Cells / mL
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68 |
63 |
44 |
59 |
58,5 |
585000
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| D9
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Count #1
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Count #2
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Count #3
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Count #4
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Average
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Cells / mL
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50 |
65 |
61 |
73 |
62,25 |
622500
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Conclusion
Related entries
Run 5: Ht31/S0 D9/D12
Same actions
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Counting hTERT cells
