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

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{|{{table}} width="800"
{|{{table}} width="800"
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span>
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Counting hTERT cells</span>
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
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<BR>
<BR>
* D9
* D9
** 1x 24-wells (25.000 cells/well (500 μL/well), 625000 cells/12.5 mL)
** 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)
** 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)
** 1x 6-wells (200.000 cells/well (2 mL/well), 1400000 cells/14 mL) --> VASP
** 8x Ø 15cm (1×10<sup>6</sup> cells/plate (15mL/plate), 3x 3×10<sup>6</sup> cells/45 mL)
** 8x Ø 15cm (1×10<sup>6</sup> cells/plate (15mL/plate), 3x 3×10<sup>6</sup> cells/45 mL) --> Anouk
<br>
<br>
* D12
* D12
** 1x 24-wells (25.000 cells/well (500 μL/well), 625.000 cells/12.5 mL)
** 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)
** 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)
** 1x 6-wells (200.000 cells/well (2 mL/well), 1.400.000 cells/14 mL) --> VASP
** 8x Ø 15cm (≤2×10<sup>6</sup> cells/plate (15 mL/plate), 3x ≤6×10<sup>6</sup> cells/45 mL)
** 8x Ø 15cm (≤2×10<sup>6</sup> cells/plate (15 mL/plate), 3x ≤6×10<sup>6</sup> cells/45 mL) --> Anouk


==Materials & Methods==
==Materials & Methods==
===Materials===
===Materials===
* 500 mL warm DMEM S<sup>+</sup>
* 500 mL warm DMEM S<sup>+</sup>
* 450 mL warm DMEM
* 10 mL PenStrep
* 3 mL Fungizone
* 50 mL FBS (HI)
* 100 mL Warm [[PBS]]
* 100 mL Warm [[PBS]]
* 4x 15 mL tubes
* 4x 15 mL tubes
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* 2x 6-wells plates
* 2x 6-wells plates
* 16x Ø 15cm petridishes
* 16x Ø 15cm petridishes
<br>
* 8 mL Trypin
* 8 mL Trypin


===Method===
===Method===
{| border="1"
====Preparation of Medium====
|+'''6-wells'''
<b><u>DMEM S<sup>+</sup></u></b>
!#
* Remove 50 mL of DMEM out of 500 mL
!<u>1</u>
* Add 50 mL FBS
!<u>2</u>
* 3 mL Fungizone
!<u>3</u>
* 10 mL PenStrep
|-
 
!A
====Collecting cells====
|
* Warm solution required in water (PBS, DMEM S<sup>+</sup>)
|
* 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
!B
* 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 <b>26</b> mL DMEM S<sup>+</sup>
{| border="1"
** Prepare plates with Name, Donor and Passage
|+'''24-wells'''
* After incubation add 5 - 10 mL DMEM S<sup>+</sup> 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
!<u>1</u>
* Remove cell suspension and add to the remaining medium in the prepared Greiner tube (26 mL + 4×1 mL trypsin = 30 mL total volume)
!<u>2</u>
 
!<u>3</u>
====[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/12| Cell counting]]====
!<u>4</u>
*Count cells using [http://en.wikipedia.org/wiki/Hemocytometer Bürker-Türk counting chamber] (See [http://upload.wikimedia.org/wikipedia/commons/f/f2/Neubauer_improved_schema.gif image 120220101])
!<u>5</u>
** Put ~10 μL in each chamber and count the cells present in 4 large squares, selected randomly from both chambers.
!<u>6</u>
* The average of cells in the 4 blocks has to be multiplied by 10<sup>4</sup> (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
!A
* Correct the amount of cells by diluting with DMEM S<sup>+</sup>
|
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* 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 (1×10<sup>6</sup> cells/plate (15mL/plate), 3x 3×10<sup>6</sup> 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 (≤2×10<sup>6</sup> cells/plate (15 mL/plate), 3x ≤6×10<sup>6</sup> cells/45 mL) --> Anouk
|-
|-
|}
|}
==Results==
==Results==
*{{todo| WHAT?}}
*{{todo| WHAT?}}
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====Run 5: Ht31/S<sup>0</sup> D9/D12====
====Run 5: Ht31/S<sup>0</sup> D9/D12====
*[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/16|Splitting cells 16March2010]]
*[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/03/16|Splitting cells 16March2010]]
 
====Same actions====
*[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/15| Preparation of Medium]]
*[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/10| Splitting cells]]
*[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/12| Cell counting]]
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->
|}
|}


__NOTOC__
__NOTOC__

Revision as of 01:26, 19 March 2010

<html><style type="text/css"> .todo { color: red } .done { color: green} </style></html>

Counting hTERT cells <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

Summary

  • Split cells
    • GIVE WOLTER 3×106 cells


  • 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)

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

  • WHAT?

Conclusion

  • DONE Ow..

Related entries

Run 5: Ht31/S0 D9/D12

Same actions


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