User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/03/19: Difference between revisions
From OpenWetWare
mNo edit summary |
m (→Results) |
||
(8 intermediate revisions by the same user not shown) | |||
Line 5: | Line 5: | ||
{|{{table}} width="800" | {|{{table}} width="800" | ||
|- | |- | ||
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> | |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> </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> </html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}} | ||
|- | |- | ||
Line 11: | Line 11: | ||
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | ||
==Summary== | ==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 & 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]] | |||
* 4x 15 mL tubes | * 4x 15 mL tubes | ||
* 8x 50 mL tubes | * 8x 50 mL tubes | ||
Line 37: | Line 27: | ||
* 2x 6-wells plates | * 2x 6-wells plates | ||
* 16x Ø 15cm petridishes | * 16x Ø 15cm petridishes | ||
* 8 mL Trypin (5x diluted) | |||
* 8 mL Trypin | |||
===Method=== | ===Method=== | ||
====Preparation of Medium==== | |||
<b><u>DMEM S<sup>+</sup></u></b> | |||
* 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<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 | |||
* 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> | |||
** Prepare plates with Name, Donor and Passage | |||
* 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 | |||
* Remove cell suspension and add to the remaining medium in the prepared Greiner tube (26 mL + 4×1 mL trypsin = 30 mL total volume) | |||
====[[User:Wilfred_J._Poppinga/Notebook/cAMP_compartmentalization/2010/02/12| Cell counting]]==== | |||
*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]) | |||
** 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 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 | |||
* Correct the amount of cells by diluting with DMEM S<sup>+</sup> | |||
<BR> | |||
{|{table} | |||
| | | | ||
* 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== | ||
| | * Cell counting (# of cells) | ||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''D12''' | |||
| align="center" style="background:#f0f0f0;"|'''Count #1''' | |||
| align="center" style="background:#f0f0f0;"|'''Count #2''' | |||
| align="center" style="background:#f0f0f0;"|'''Count #3''' | |||
| align="center" style="background:#f0f0f0;"|'''Count #4''' | |||
| align="center" style="background:#f0f0f0;"|'''Average''' | |||
| | | align="center" style="background:#f0f0f0;"|'''Cells / mL''' | ||
| | |||
| | |||
| | |||
| | |||
| | |||
|- | |- | ||
| ||68||63||44||59||58,5||585000 | |||
| | |||
| | |||
| | |||
| | |||
| | |||
| | |||
|- | |- | ||
| align="center" style="background:#f0f0f0;"|'''D9''' | |||
| | | align="center" style="background:#f0f0f0;"|'''Count #1''' | ||
| | | align="center" style="background:#f0f0f0;"|'''Count #2''' | ||
| | | align="center" style="background:#f0f0f0;"|'''Count #3''' | ||
| | | align="center" style="background:#f0f0f0;"|'''Count #4''' | ||
| | | align="center" style="background:#f0f0f0;"|'''Average''' | ||
| | | align="center" style="background:#f0f0f0;"|'''Cells / mL''' | ||
|- | |- | ||
| ||50||65||61||73||62,25||622500 | |||
| | |||
| | |||
| | |||
| | |||
| | |||
| | |||
|- | |- | ||
|} | |} | ||
{| | <BR> | ||
| | * Preparing cell dilutions | ||
|# | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''D12''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''24-wells''' | |||
| align="center" style="background:#f0f0f0;"|'''10 cm''' | |||
| align="center" style="background:#f0f0f0;"|'''6-wells''' | |||
| align="center" style="background:#f0f0f0;"|'''15 cm''' | |||
|- | |- | ||
| Cells||585000||625000||1500000||1400000||6000000 | |||
| | |||
| | |||
| | |||
| | |||
| | |||
| | |||
| | |||
| | |||
| | |||
| | |||
| | |||
|- | |- | ||
| align="center" style="background:#f00000;"| mL Cells | |||
| | | align="center" style="background:#f00000;"|1 | ||
| | | align="center" style="background:#f00000;"|1,07 | ||
| | | align="center" style="background:#f00000;"|2,56 | ||
| | | align="center" style="background:#f00000;"|2,39 | ||
| | | align="center" style="background:#f00000;"|10,26 | ||
| | |||
| | |||
| | |||
| | |||
| | |||
| | |||
| | |||
|- | |- | ||
| align="center" style="background:#f00000;"| mL S+|| | |||
| | | align="center" style="background:#f00000;"|11,43 | ||
| | | align="center" style="background:#f00000;"|17,44 | ||
| | | align="center" style="background:#f00000;"|11,61 | ||
| | | align="center" style="background:#f00000;"|34,74 | ||
| | |||
| | |||
| | |||
| | |||
| | |||
| | |||
| | |||
| | |||
|- | |- | ||
| SUM||1,00||12,50||20,00||14,00||45,00 | |||
| | |||
| | |||
| | |||
| | |||
| | |||
| | |||
| | |||
| | |||
| | |||
| | |||
| | |||
|- | |- | ||
|} | |||
| | {| {{table}} | ||
| | | align="center" style="background:#f0f0f0;"|'''D9''' | ||
| | | align="center" style="background:#f0f0f0;"|'''''' | ||
| | | align="center" style="background:#f0f0f0;"|'''24-wells''' | ||
| | | align="center" style="background:#f0f0f0;"|'''10 cm''' | ||
| | | align="center" style="background:#f0f0f0;"|'''6-wells''' | ||
| | | align="center" style="background:#f0f0f0;"|'''15 cm''' | ||
| | |||
| | |||
| | |||
| | |||
| | |||
|- | |- | ||
| Cells||622500||625000||1500000||1400000||3000000 | |||
| | |||
| | |||
| | |||
| | |||
| | |||
| | |||
| | |||
| | |||
| | |||
| | |||
| | |||
|- | |- | ||
| align="center" style="background:#f00000;"| mL Cells | |||
| | | align="center" style="background:#f00000;"|1 | ||
| | | align="center" style="background:#f00000;"|1,00 | ||
| | | align="center" style="background:#f00000;"|2,41 | ||
| | | align="center" style="background:#f00000;"|2,25 | ||
| | | align="center" style="background:#f00000;"|4,82 | ||
| | |||
| | |||
| | |||
| | |||
| | |||
| | |||
| | |||
|- | |- | ||
| align="center" style="background:#f00000;"| mL S+ | |||
| | || | ||
| | | align="center" style="background:#f00000;"|11,50 | ||
| | | align="center" style="background:#f00000;"|17,59 | ||
| | | align="center" style="background:#f00000;"|11,75 | ||
| | | align="center" style="background:#f00000;"|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. | |||
* | |||
==Related entries== | |||
===Run 5: Ht31/S<sup>0</sup> D9/D12=== | |||
*[[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 06:04, 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> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
SummaryTo 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 & MethodsMaterials
MethodPreparation of MediumDMEM S+
Collecting cells
Cell counting
Results
Related entriesRun 5: Ht31/S0 D9/D12Same actions |