An efficient dissociation protocol for generation of single cell suspension from zebrafish embryos and larvae.

Zebrafish (Danio rerio) has emerged as a powerful animal model to study developmental processes and human diseases. The introduction of CRISPR/Cas9 as a genome editing tool allowed the generation of genetic mutants with high-throughput (Varshney et al., 2015) and has opened the possibility to understand gene function not only during embryonic stages but also in larval stages. Therefore, there is an increasing need to optimize methods for embryo and larvae dissociation that allow the generation of single cell suspension for fluorescence-activated cell sorting (FACS), RNA extraction and single cell RNA-sequencing.

Here we present a quick and efficient protocol for the dissociation of zebrafish embryos and larvae (2–16 days post fertilization (dpf), Fig. 1). This protocol was built empirically and optimized based on a method protocol from Gallardo and Behra (2013) [1].

Fig. 1

Visual representation of the 3-Step protocol for zebrafish embryos and larvae dissociation.

The protocol is divided in three steps:

Experimental set up (∼10 min)

Dissociation with trypsin and collagenase (∼10–15 min)

Preparation of single cell suspension (∼15 min)

This protocol has been optimized in order to be used for FACS, RNA extraction (for RNA sequencing) and single-cell RNA sequencing.

Specifications Table

Method details

Experimental set-up

Reagent preparation

Resuspend the collagenase in PBS 1X to prepare a 100 mg/ml stock. Aliquot (40 μl each), store at −20 °C. Use a fresh aliquot for each experiment.

Prepare DMEM-10%FBS (make fresh every time) and equilibrate at ∼30 °C.

Prepare the Dissociation Mix* and keep at 30 °C. Each tube requires 500 μl of Dissociation Mix.

*Dissociation Mix

480 μl 0.25% trypsin-EDTA + 20 μl Collagenase 100 mg/ml (for embryos 2–4 dpf)

OR

460 μl 0.25% trypsin-EDTA + 40 μl Collagenase 100 mg/ml (for 5–16 dpf embryos)

Zebrafish preparation

Collect zebrafish embryos or larvae at the desired stage. Euthanize embryos or larvae with Tricaine and quickly proceed to the next step. CRITICAL: Do not freeze samples.

Transfer zebrafish embryos or larvae into 1.5 ml microcentrifuge tube

∼ 30 embryos/tube for 2–4 dpf

∼ 20 embryos/tube 4–10 dpf

∼ 5 embryos/tube 10–16 dpf

Wash 2X in 1 ml PBS 1X

Dissociation with trypsin and collagenase

Add dissociation mix

Remove PBS 1X

Add 500 μl of Dissociation Mix* (pre-heated at 30 °C) to each 1.5 ml microcentrifuge tube

Mechanically homogenize

Mechanically dissociate the embryos via harsh pipetting (use a P1000 and then P200). Interval ∼30 s pipetting and ∼30 s in heat-block at 30 °C until tissue is no longer visible (5–10 min).

NOTE: once fully homogenized, the mixture will appear lighter in color.

Stop dissociation

Add 800 μl of DMEM-10%FBS to the Dissociation Mix

Mix and centrifuge 5 min at 700 g at room temperature (RT)

NOTE: after centrifugation the pellet should be clearly visible.

Preparation of single cell suspension

Wash the pellet

Discard the supernatant

Resuspend the pellet in 1 ml PBS 1X

Centrifuge 5 min at 700 g at RT

Filter single cell suspension

Discard the supernatant and resuspend in 0.5–1 ml of DMEM-10%FBS

NOTE: If multiple samples need to be pooled for downstream applications it can be done at this resuspension step. Resuspend pellet 1 with DMEM-10%FBS and use the resuspension to resuspend subsequent samples (consider 1 ml for up to 3 samples) and then proceed to the next step.

Filter through 70 μm nylon mesh into 50 ml conical tube

Wash the filter with 500 μl of DMEM-10%FBS

Transfer the cell suspension

Transfer the single cell suspension into a new tube and proceed with downstream applications.

Optional Step: If a smaller volume is required cells can be centrifuge 5 min at 700 g at RT and resuspended in DMEM-10%FBS or a solution that downstream applications require.

CRITICAL: Keep the cell suspension at room temperature and do not store on ice.

Subject Area	Biochemistry, Genetics and Molecular Biology
More specific subject area:	Developmental biology, zebrafish
Method name:	Zebrafish embryo, larvae dissociation to single cell suspension
Name and reference of original method	Our dissociation protocol was built empirically and optimized based on a method protocol from Gallardo et al.Gallardo VE, Behra M. Fluorescent activated cell sorting (FACS) combined with gene expression microarrays for transcription enrichment profiling of zebrafish lateral line cells. Methods. 2013;62(3):226-231.
Resource availability	Reagents:• Collagenase from Clostridium histolyticum (Sigma #C9891) • PBS 1X without Calcium and Magnesium (e.g. Corning #21-040-CV) • DMEM 1X with 4.5 g/L glucose, L-glutamine & sodium pyruvate (Corning 10-013-CV) • Fetal Bovine Serum (Fisher Scientific #SH30071.03HI) • 0.25% trypsin-EDTA (Gibco #25200-056) • Pharmaceutical grade buffered Tricaine methane sulfonate (MS-222) Materials:• Heat block set at 30 °C • 1.5 ml Microcentrifuge tubes (clear) • P1000 and P200 pipette tips • Centrifuge • Cell strainer, 70 μm mesh (e.g. FALCON #352350) • 50 ml Conical tubes