TITLE: Protocol to process Gastruloids for FACS (reporters only) 
AUTHORS: Stefano Vianello, Tania Hübscher, Giuliana Rossi, Matthias Lutolf

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol to process reporter Gastruloids for FACS analysis of their endogenous reporters.</div></div>

[STEPS]
?. [Gastruloid collection]
Once all Gastruloids have settled to the bottom of the tube, aspirate out the supernatant (N2B27 medium carried over with each Gastruloids)
?. [Gastruloid collection]
Resuspend the Gastruloids in 5mL PBS-/-, to wash away traces of N2B27
?. [Gastruloid collection]
Once all Gastruloids have re-settled to the bottom of the tube, aspirate out the PBS-/-.Gastruloids are ready to be digested.
?. [Gastruloid digestion]
Digest the Gastruloids by adding 1mL Digestion Solution (3mg/mL Collagenase IV, 4mg/mL Dispase, 100ug/mL DNAseI, in PBS-/-),  ,  min
[(waterbath)]
?. [Gastruloid digestion]
After the 4min incubation has elapsed, use a P1000 coated in Staining Buffer to try to mechanically disrupt the Gastruloids
?. [Gastruloid digestion]
Place the tube back again at  ,  min; to complete the digestion
[(waterbath)]
?. [Gastruloid digestion]
After the last 4min of  incubation have elapsed, use a P1000 coated in Staining Buffer (see recipe in "Materials" section)  to mechanically disrupt the Gastruloids. Gastruloids should break easily and give rise to a single cell suspension.
?. [Cell filtering]
Prewet the blue cap of a filter-FACS tube (e.g. CAT#352235, Falcon/Corning), by pipetting 1mL of Staining Buffer through it.
This step is very imprtant. Not prewetting the filter can lead to loss of a high number of cells at the filtering step.
?. [Cell filtering]
Working Filter your cell suspension (digested Gastruloids) through the cap of the FACS tube
on ice
?. [Cell filtering]
Wash the filter by passing 1mL more of DIgestion Solution through it.
on ice
?. [Cell filtering]
Transfer the contents of the FACS tube to a clean 15mL Falcon tube.
on ice
?. [Cell filtering]
Collect any leftover cells by flushing the old FACS tube with 2mL Staining Buffer, and transferring in the 15mL Falcon tube with the rest of the cells
on ice
?. [Cell filtering]
Add 5mL more Staining buffer into the tube Falcon tube with your cells suspension, to completely stop the digestion reaction. This tube now contains a filtered, single-cell suspension of your sample for FACS.
on ice
?. [Cell filtering]
Spin down the cell suspension,  ,  min
Centrifuge: 200 34, 4 10
?. [DAPI staining and fixation]
For each sample, prepare 500uL of 0.2ug/mL DAPI in Staining Buffer
From a stock solution of DAPI 1mg/mL, use 1:5000. E.g. add 0.1uL DAPI in 500uL Staining Buffer
?. [DAPI staining and fixation]
Resuspend the cell pellet in the DAPI solution, and incubate  min,
on ice
on ice
?. [DAPI staining and fixation]
Spin down the cell suspension,  ,  min
Centrifuge: 200 34, 4 10
?. [DAPI staining and fixation]
FIxation:Resuspend the DAPI-stained cell pellet in 2mL 1%PFA,
on ice
?. [DAPI staining and fixation]
Spin down the cell suspension,  ,  min
Centrifuge: 200 34, 4 10
?. [DAPI staining and fixation]
Resuspend in 300uL Staining Buffer and transfer to a clean FACS tube pre-coated with Staining Buffer
?. [Cell collection]
For samples that come from 2D culture (e.g. 2D-grown reporter cells as negative controls, or 2D-grown single-color cells as positive controls):aspirate out the medium from the culture dishadd 3mL PBS-/- to wash away any trace of medium, and aspirate it out againAdd 500uL Accutase, to detach the cells,   min,   Transfer the detached cells +Accutase to a clean Falcon tubeAdd ~5mL 10% Serum to stop the reactionAdd 500uL Accutase, to detach the cells,  min, Transfer the detached cells +Accutase to a clean Falcon tubeAdd ~5mL 10% Serum to stop the reactionSpin down the cell suspension,  ,  minThe cells are ready to be processed just as any Gastruloid sample (proceed to the next step)
0 Room temperature
Centrifuge: 200 34, 4 10
?. [Gastruloid collection]
Harvest Gastruloid from each well of  a 96 well plate and collect them in a 15mL Falcon tube (pooled by timepoint or condition)
?. [Processing/Storage]
Cells can be kept in Staining Buffer,  , in the dark (wrapped in alluminium foil) for at least a week  (e.g. while waiting to collect samples from later on timepoints)
4 °C
?. [Processing/Storage]
When ready for FACS,  ,distribute your cells in 300uL per FACS tube and proceed for sorting/analysis.
on ice
?. [Beads samples]
If using beads as positive references, prepare bead tubes just before FACS processing. For GFP BrightComp eBeads™ Compensation Bead Kit (Invitrogen/Thermo Fisher Scientific, CAT#A10514):vortex vial for  sDispense 1 drop into a clean FACS tubeResuspend in 1mL Staining Buffer