Tissue dissocication of zebrafish embryo with chorion

This protocol enables the dissociation of whole zebrafish embryo with chorion, i.e. before 3-days-post-fertilization, for downstream cell sortiong or DNA/RNA extraction.

Experimental Procedures

1. Zebrafish dechorination

Dechorionate the embryos with pronase: Add 1mL stock pronase solution to 15 ml fish water with embryos and keep in room temperature for 15-20 minutes. Rotate gently to release embryos from chorion.  If necessary, you may use forceps afterward to gently pull them from the softened chorion. Grabbing the chorion and pulling to the surface should be enough.  Rinse embryos gently with fish water.

1. Zebrafish embryo dissociation
2. Rinse embryos 3 X 5 minutes in PBS @ RT on shaker, after which all embryos will be transfer gently to a 1.5mL eppi-tube.
3. Disassociate embryos in eppi-tube in 1 ml of PBS with Micropestle 5-6 times with twisting motion. 
4. Centrifuge @ 500g for 5 minutes @ RT.
5. Discard Supernatant 
6. Resuspend in 1 ml of 0.25% Trypsin +EDTA.
7. Incubate @ 33° C for about 30 minutes.  Pipette every 5-10 minutes to keep it from clumping. While incubating, you can coat a 50 ml conical tube with 2-3 mls of 5% FBS in 1X PBS and keep this on ice.  This keeps the cells from sticking to the tube when you filter them.
8. Centrifuge cells @ 500g for 5 minutes @ 4° C.
9. Wash 1 x 5 minutes in ice cold PBS/FBS(5%) on the 4° C shaker.  After wash re-pellet (step 7). 
10. Resuspend in 500 µl of cold 5% FBS in 1x PBS. 
11. Collecting cells for FACS
    1. Filter through a 40 µm cell strainer (Fisher 08-771-1) into the empty coated 50 ml conical tube from step 6.  (add all the resuspended cells in 9) to the filter)
    2. Rinse eppi with 500 µl of FBS/PBS and filter. (rinse the epi tube with 500uL    FBS/PBS). Centrifuge at 500g for 2min.
    3. Wash filter with 500 µl of FBS/PBS. Centrifuge at 750g for 7 min.
    4. Resuspend the cell pellete with FBS/PBS. After cell counting, go for sorting (the ideal concentration of cells suspension for sorting should be between 5 x 10⁶ and 30 x 10⁶/ml.

Materials

• Fetal Bovine Serum (FBS) for regular cell culture
• Eppendorf^® Micropestle
• Phosphate Buffered Saline, PBS (pH=7.2) for cell culture (e.g. ,Gibco 20012027)

              PBS/FBS (5%, v/v))

• 0.25% Trypsin+EDTA (for cell culture) (e.g., Gibco 25200056)
• 40 µm cell strainer (e.g. Falcon 352340)
• Pronase from Streptomyces griseus (Roche 10165921001)

Make a 10 mg/ml stock solution (10 ml) of Pronase and add 1 ml each into ten  15 ml falcon tubes. These can be frozen at -20ºC until needed, avoid repeated thawing and freezing.