Imaging of lipid droplets, nuclei and cytoplasm in the 96-well format (Thiele lab)

From LipidomicsWiki

96 – well plate – imaging of lipid droplets, cytoplasm and nuclei

This protocol is used for automated, high-content image acquisition by the OPERA from Evotec.
Depending on the cell line, multi-well plate and microscope used, staining conditions for optimal images may vary.

Additionally, dye concentrations may need to be adapted depending on the question under study.
As an example, cells supplemented with during cell culture need lower BODIPY493 concentrations
compared to starving (using delipidated FCS) conditions during cell culture.

Fixation:
- remove culture medium
- wash cells 1 x with 100 µl/well 1x D-PBS
- fix cells by adding 100 µl/well 1x D-PBS + 5 % Formaldehyde
- incubate 1 hour at RT
- remove fixation solution
- wash cells 1 x with 100 µl/well 1x D-PBS
- add 100 µl/well 1x D-PBS
- optional: store the cells at 4 degree Celsius
- OR continue immediately with the Staining procedure

Staining:
- Wash cells 2x with 100 µl/well water
- add dye solution (80 – 100 µl/well):
o    DAPI: 1 µg/ml
o    Syto42 (Invitrogen): 2 - 3 µM
o    Bodipy493/503 (Invitrogen): 0.5-2.5 µg/ml
- incubate in the dark for 1 hr, RT
- wash 1x with 100 µl/well water
- quick wash 1x with 100 µl/well water + 1.5 % fat free BSA
Note: 1.5 % fat free BSA is added to reduce the Bodipy background. If it is added too long it will disturb the procedure!!!
- wash 2x with 100 µl/well D-PBS
- add 100 µl D-PBS per well and cover the plate with ‘silver tape’

proceed to image acquisition