Dissociation and FACS of embryonic tissue

FACS of e11.5 foreguts

Solutions

10% FBS in PBS

Need 500ul/collection tube

FACS buffer (20ml):

10% FBS - 2ml

2mM EDTA - 80ul

HBSS - 18ml

Sytox solution (pacific blue):

*sytox located in nuclear counterstains box in -20°C

1:8000 in FACS buffer

Antibodies:

EpCam-PerCP-Cy5.5 (BioLegend), use at 1:100 in FACS buffer, stain in 100-200ul volume

RLT + bME

5ml RLT from Qiagen RNeasy micro kit + 50ul beta-Mercaptoethanol (can store at 4°C and use w/in 2 weeks)

Protocol

1. Dissect out whole foreguts into 500ul cold PBS w/ 10% FBS in 2ml LoBind eppendorf tube.
   1. If pooling foreguts, combine up to 10 foreguts/tube.
   2. If processing foreguts individually, put each foregut into its own tube.
2. Remove PBS w/ FBS thoroughly (use p20 to remove remaining solution from tube w/out sucking up foreguts)
3.  Add 700ul TrypLE/tube for 5-10 foreguts, 500ul TrypLE for 1 foregut.
4. Incubate tube in 37°C water bath for 5 minutes.
5. Begin triturating with p1000 every 2-5 minutes until tissue is completely dissolved. Put tubes at 37°C between trituration.
   1. Be very careful not to let foregut stick to your pipette tip! This can be helped by:
      1. Pulling up and dispensing 1ml fresh TrypLE through pipette tip before trituration to “wet” pipette tip.
      2. Never sucking up >500ul during trituration. 
      3. Rapid trituration to move around liquid, not forceful trituration to suck up and dispense foreguts.
6. Add 1ml FACS buffer
7. Take 10% of total volume and transfer to a new tube to use as unstained control.
8. Spin down cells at 600xg, 5min.
9. Resuspend in 100ul antibody solution or FACS buffer for unstained ctrl.
10. Incubate on ice for 20-30min, with occasional flicking (protect from light).
11. “Wash” by adding 1.5ml FACS buffer/tube.
12. Filter through strainer to FACS tube.
13. Wash Eppendorf tube and filter with 500ul FACS buffer.
14. Pellet cells at 600xg, 5min in the TC room centrifuge in the 15ml conical bucket. The tubes will drop in, you will need to retrieve them w/ clean forceps.
15. Resuspend in 300ul FACS buffer + sytox.
16. Take tubes to FACS on ice, protected from light.

To bring to FACS

1. Extra FACS buffer
2. Dry ice
3. Eppendorf tubes with 300ul RLT + bME labelled for collection.
4. Samples

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