Date : 2022-07-04

Author : V Chong-Morrison

Dissociation of zebrafish larvae for FACS - micro version for bulk/plate-based single cell applications

Introduction

This protocol has been tested on dissected neuroepithelium tissue (aiming for pineal organ) of ~30-32 hpf larvae to FAC-sort GFP+ pineal cells with a 100 µm nozzle. For negative gating, ~10 whole wild type embryos of similar stage is sufficient.

Materials

• RNase-free pipette tips, low-binding/retention grade, for cell work
• RNase-free pipette tips and micro-centrifuge tubes, normal retention i.e. not low-binding, for buffers and reagents
• Protein LoBind® Tubes, 1.5 ml (0030108116, Eppendorf)
• pluriStrainer Mini 40 µm cell strainers (43-10040-60, pluriSelect)
• 12x75 mm 5 ml polystyrene sterile round tubes with snap cap (734-0436, Falcon)
• DNaseI (10104159001, Sigma-Aldrich)
• Papain, 10 mg/ml i.e. 0.3 U/µl (10108014001, Sigma-Aldrich)
• ROCK inhibitor a.k.a. Y-27632 dihydrochloride (A3008, Generon UK)
• 10X HBSS, no calcium, no magnesium, no phenol red (14185052, ThermoFisher)
• Bovine Serum Albumin, heat shock fraction, protease free, essentially globulin free, pH 7, >=98% (A3059, Sigma-Aldrich)
• 1M HEPES solution (15630056, ThermoFisher)
• eBioscience Fixable Viability Dye eFluor 780 (65-0865-14, ThermoFisher)

Procedure

Pre-flight

Ensure following reagents are prepared in advance and stored appropriately.

• ROCK inhibitor; resuspend 10mg with 1 ml of DMSO to concentration of 10 mg/ml. Store in -20C.
• DNaseI; resuspend 50mg/100mg with 5 ml/10ml of nuclease-free water to final concentration of 10 mg/ml. Filter sterilise (0.22 µm) and store 1 ml aliquots at 4C for couple of days or -20C for long-term.
• eFluor 780 dye; dilute a small aliquot 1 in 10 with DMSO and store at -20C. Neat stock should be stored in -80C for long-term.
• Autoclave MilliQ DEPC water for making buffers and solutions, or use commercial UltraPure DNase/RNase-free distilled water.

Prep

1. Prepare Hank’s solution (blocking isotonic solution for two samples - one experimental, one negative. Always have a negative sample for FACS gating!).

   Hank’s solution

   Component	Volume/amount	Final conc.
   10X HBSS	1.5 ml	1X
   BSA	0.0375 g	2.5 mg/ml
   1M HEPES	150 µl	10 mM
   Water	up to 15 ml	-

2. Filter-sterilise (0.22 µm) into a Falcon tube. This will now be used as follows (approximate volumes).

   Hank’s solution - volumes

   Component	Volume/amount
   Dissection	10 ml per plate
   Tissue collection	X µl (10 µl per tube)
   Dissociation	0.5 ml (2 x 250 µl)
   Stop Diss.	1.2 ml (2 x 600 µl)
   Cells collection	X µl (100 µl per tube)

   Leftover ~4 ml for pre-coating tips during dissociation process.

Steps

1. Set heat-block to 37C. Cool tabletop micro-centrifuge down to 4C.
2. If required, anaesthetise larvae and dissect tissues in Petri dish filled with 10 mL filtered Hank’s plus tricaine.
3. Prepare tissue collection tubes for experimental and negative samples. Pipette 250 µl filtered Hank’s into a Protein LoBind® tube, mark level of liquid with a marker, then remove 240 µl leaving behind 10 µl filtered Hank’s. Place on ice.
4. Using a glass Pasteur pipette, collect dissected tissue into prepared tubes kept cool on ice throughout.
5. Collect tissue within 30-40 minutes to maximise viability.
6. Top back up with filtered Hank’s to 250 µl mark.

7. Add 20 µl Papain, 0.3 µl ROCK Inhibitor and 30 µl DNaseI to commence dissociation.

   Dissociation reaction

   Component	Volume/amount	Final conc.
   Papain	20 µl	0.02 U/µl
   ROCK inhibitor	0.3 µl	10 µg/ml
   DNaseI	30 µl	1 mg/ml
   Hank’s (filtered)	up to 300 µl	-

8. Incubate at 37C. Every 5 minutes, triturate sample 20-30 times in solution against the tube wall using a p200 (set to 100 µl) low-binding tip. Tip: Pre-coat the tip with filtered Hank’s before triturating.
9. Repeat step 4 until sample is dissociated i.e. no pieces of tissue left.
10. Stop dissociation by adding 600 µl filtered Hank’s.
11. Place a 40 µm cell strainer into a fresh Protein LoBind® tube.
12. Pre-coat a P1000 tip (set to 500 µl) with filtered Hank’s before gently resuspending the cell suspension and transfer everything onto the cell strainer.
13. With cell strainer still attached, centrifuge at 500 g/rcf at 4C for 5 minutes.
14. Bulk cell applications. Prepare normal retention tubes (if planning to pellet cells) for FACS collection - 100 µl filtered Hank’s per tube.
15. Discard cell strainer and gently remove supernatant until approximately 500 µl remain.
16. Add 0.2 ul of 1:10 diluted eFluor 780 dye.
17. Resuspend the cell pellet gently using a fresh pre-coated p200 (set to 100 µl) low-binding tip and transfer to a 5 ml polystyrene round tube.
18. Place on ice and proceed with FACS (about 30 minutes to sort entire sample).
19. Bulk cell applications. After FACS, pellet collected cells 500 g/rcf at 4C for 5 minutes in cooled tabletop micro-centrifuge. Very carefully, remove the supernatant and proceed with downstream applications.

Appendix

Examples of downstream applications

• RNA extraction - resuspend pelleted cells in 100 µl Lysis Buffer (RNAqueous™-Micro Total RNA Isolation Kit, AM1931, ThermoFisher) and proceed with RNA extraction.
• ATAC-seq - protocol available separately.

Evaluating dissociations

If desired, success of dissociations can be evaluated in trial runs using Trypan Blue viable cell counting with a haemocytometer. A quick Internet search will bring up several standard protocols. Here is an example.

Protocol adjustments for different stages/tissue

Ensure the amount of tissue is roughly the same as the protocol. The drawback to this approach is that the older the embryos, the fewer you can fit in - if the target population is not a rare cell type this may not be a problem.

For younger embryos, the protocol remains the same. However, if younger than ~bud stage, a deyolking step may be needed prior to dissociation as excess yolk may inhibit enzyme (papain) activity.