TITLE: Dissociation of EBs using Worthington Kit
AUTHORS: Niraj Sawarkar, Regine Tipon, Gist Croft

[DESCRIPTION]
Dissociation of EBs using papain

[STEPS]
SECTION: Reagent prep
1. Add 32 ml of EBSS to the albumin ovomucoid inhibitor
pipet to  dissolve 
SECTION: Reagent prep
5. Bring CMF-PBS + Glucose (1 X PBS Ca2+/Mg2+
Free) + 12.5 ml of 1M Glucose (Ratio of 1:40) 
SECTION: Reagent prep
2. Add 5 ml of EBSS to a papain vial. Place in a 37°C water bath for 10-15 min mins until the papain is completely dissolved and the solution appears clear. Use freshly made papain for each dissociation.
SECTION: Reagent prep
3. Add 500 µls of EBSS to a DNase vial. Mix gently – DNase is sensitive to shear denaturation.
SECTION: Reagent prep
4. Thaw 1 aliquot  of 10mg/nl DNASe 
SECTION: Reagent prep
6. Bring CTWM to the hood (1 X PBS Ca2+/Mg2+
Free) + 12.5 ml of 1M Glucose (Ratio of 1:40) + 12.5ml of 4% dialyzed BSA
(Ratio 1:40) + 5ml of N2 + 10ml B27 +1 ml of MgCl2 (stock 2mM; Ratio
1:500) + 500ml of EDTA

SECTION: Reagent prep
7. Prepare 50ml of CTWM plus ODD mix [1X (15ml) CTWM+ODD 2X (30ml)]+Worthignton DNAse 300ul (10mg/ml DNase)

 
  

 
SECTION: Dissociation protocol (optimized)
8. Collect EBs and add to a 15ml tube,
SECTION: Dissociation protocol (optimized)
9. Allow to settle (2 min) if required, spin at 30g for 3 min, and aspirate medium (careful not to aspirate EBs!)
SECTION: Dissociation protocol (optimized)
10. Add 10m of CMF-PBS+Glucose; allow EBs to settle and aspirate medium
SECTION: Dissociation protocol (optimized)
11. Prepare Papain DNAse solution for all samples by adding 100ul DNAse/ 1ml Papain
SECTION: Dissociation protocol (optimized)
12. Add 1ml Papain/DNAse to / ~10M cells or ensure that all the EBs are completely submerged within the Papain/DNAse mixture.
SECTION: Dissociation protocol (optimized)
13. Incubate the vial containing the tissue at 37°C with periodic swirling (a rocker platform is ideal) for 30 to 90 min, depending on age and size of Ebs (day 20 Ebs for 30 min or less, day 33-120 for 90 min). Keep shaking the tube every 15 to 20 minutes to ensure uniform dissociation.
SECTION: Dissociation protocol (optimized)
14. After incubation is complete, spray the tube with ethanol and return it to the hood.
SECTION: Dissociation protocol (optimized)
15. Add 8 ml CTWM (Complete Trituration Wash medium)+ODD mixture—
SECTION: Dissociation protocol (optimized)
16. Spin 300G x 3min
SECTION: Dissociation protocol (optimized)
17. Aspirate supernatant
SECTION: Dissociation protocol (optimized)
18. Resuspend with 1ml of CTWM+ODD and Triturate 20 passes with p1000
SECTION: Dissociation protocol (optimized)
19. Add 9 ml CTWM+ODD and mix
SECTION: Dissociation protocol (optimized)
20. Important if the triturated EBs start looking sticky and/or thread-like, quickly add 10-15 ul of DNAse to the tube and triturate until a homogeneous mixture is obtained.
SECTION: Dissociation protocol (optimized)
21. Spin 500G x 3min and aspirate supernatant
SECTION: Dissociation protocol (optimized)
22. If there are still suspended particles throughout the media, add 10-15 ul of DNAse and resuspend, followed by spin once again.
SECTION: Dissociation protocol (optimized)
23. Add 10 ml of seeding media and aliquot to count cells.
SECTION: Dissociation protocol (optimized)
24. Spin 500Gx3min
SECTION: Dissociation protocol (optimized)
25. Aspirate sup and resuspend to high-density stock in seeding medium (10M cells/ml, 5M cells/ml, 2M cells ml as appropriate for total cell number in
SECTION: Dissociation protocol (optimized)
26. Dilute for seeding stock
SECTION: Dissociation protocol (optimized)
27. Seed 96 wp wells at high density