HTP Image analysis of LD size in differentiated adipocytes
From LipidomicsWiki
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Current revision (13:26, 16 April 2009) (view source) |
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| - | General | + | = General = |
| - | <br> | + | use black plates (384 or 96 well plates)<br>This protocol is designed for fixed cells; all dyes can be adapted for live cells |
| - | + | <br> | |
| - | <br> | + | =Fixation= |
| + | Remove culture medium<br>Wash cells 1 x with 100 µl/well 1 x PBS<br>Fix cells by adding 100 µl/well 1 x PBS + 4 % FA<br>Incubate 1 hour at RT or o/n at 4°C<br>Remove fixation solution<br>Wash cells 1 x with 100 µl/well 1 x PBS<br>Add 100 µl/well 1 x PBS<br>• optional: store the cells at 4 degree Celsius OR continue immediately with the Staining procedure | ||
| - | + | <br> | |
| + | |||
| + | =Staining= | ||
| + | Wash cells 2 x with water (100 µl/well)<br>Add dye solution (80 – 100 µl/well):<br>dye solution in water:<br>o Hoechst : 4 µM (stock 2 mM)<br>o Syto60 : 5 µM (stock 5 mM)<br>o Bodipy : 1 µM (stock 5 mM)<br>Incubate in the dark for 1 hr, RT <br>Wash 1 x with water (100 µl/well)<br>Wash each well for 2 min with water + 1.5 % fat free BSA (100 µl/well) | ||
Note: 1.5 % fat free BSA is added to reduce the Bodipy background. If it is added too long it will destroy the LDs!!! Wash exactly for 2 min, if you have too many wells process in batches | Note: 1.5 % fat free BSA is added to reduce the Bodipy background. If it is added too long it will destroy the LDs!!! Wash exactly for 2 min, if you have too many wells process in batches | ||
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Wash 2 x with PBS (100 µl/well)<br>Add 100 µl PBS per well, image acquisition | Wash 2 x with PBS (100 µl/well)<br>Add 100 µl PBS per well, image acquisition | ||
| - | <br> | + | <br> |
| - | + | ||
| - | + | ||
| - | Image | + | =Image Acquisition= |
| + | Use Cellworx machine<br>Use Hoechst to calculate z plane, and record the best zplane<br>Do not change the gain or offsets | ||
| - | adaipted protocol from Dresden<br><br> | + | =Image Analysis= |
| + | Run on Cellprofiler using the LDDetection Pipeline<br><br> | ||
| + | adaipted protocol from Dresden<br><br> | ||
[[Category:Lipid_imaging]] | [[Category:Lipid_imaging]] | ||
Current revision
Contents |
General
use black plates (384 or 96 well plates)
This protocol is designed for fixed cells; all dyes can be adapted for live cells
Fixation
Remove culture medium
Wash cells 1 x with 100 µl/well 1 x PBS
Fix cells by adding 100 µl/well 1 x PBS + 4 % FA
Incubate 1 hour at RT or o/n at 4°C
Remove fixation solution
Wash cells 1 x with 100 µl/well 1 x PBS
Add 100 µl/well 1 x PBS
• optional: store the cells at 4 degree Celsius OR continue immediately with the Staining procedure
Staining
Wash cells 2 x with water (100 µl/well)
Add dye solution (80 – 100 µl/well):
dye solution in water:
o Hoechst : 4 µM (stock 2 mM)
o Syto60 : 5 µM (stock 5 mM)
o Bodipy : 1 µM (stock 5 mM)
Incubate in the dark for 1 hr, RT
Wash 1 x with water (100 µl/well)
Wash each well for 2 min with water + 1.5 % fat free BSA (100 µl/well)
Note: 1.5 % fat free BSA is added to reduce the Bodipy background. If it is added too long it will destroy the LDs!!! Wash exactly for 2 min, if you have too many wells process in batches
Wash 2 x with PBS (100 µl/well)
Add 100 µl PBS per well, image acquisition
Image Acquisition
Use Cellworx machine
Use Hoechst to calculate z plane, and record the best zplane
Do not change the gain or offsets
Image Analysis
Run on Cellprofiler using the LDDetection Pipeline
adaipted protocol from Dresden
