HTP Image analysis of LD size in differentiated adipocytes

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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

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