Table of Contents

• Table of Contents
• Setup
  • Reagents
  • Materials
  • Buffers
    • Artificial seawater (ASW) - CSHL Protocols
    • ASW from dry salts
    • Dissociation Mix
      • (for embryos 2–4 dpf)
      • (for embryos 5–16 dpf)
  • Reagent Prep
• Protocol
  • Speciment Prep
  • Enzymatic Dissociation
  • Preparation of single cell suspension
• Alternative mechanical dissociation

Setup

Reagents

• Collagenase from Clostridium histolyticum (Sigma #C9891)-
• PBS 1X without Calcium and Magnesium (e.g. Corning #21-040-CV)
  • TODO: Replace with ASW or equivalent?
• DMEM 1X with 4.5 g/L glucose, L-glutamine & sodium pyruvate (Corning 10-013-CV)
  • TODO: Replace with ASW or equivalent?
• Fetal Bovine Serum (Fisher Scientific #SH30071.03HI) (to stop trypsin)
• 0.25% trypsin-EDTA (Gibco #25200-056)
  • TODO: Replace with TryplE or equivalent?
• Pharmaceutical grade buffered Tricaine methane sulfonate (MS-222)
  • TODO:This is likely EtOH instead for cephs.

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

Buffers

Artificial seawater (ASW) - CSHL Protocols

• pH 7.4
• Filter sterilize and store at 4°C

ASW from dry salts

• pH 7.4
• Filter sterilize and store at 4°C

Dissociation Mix

(for embryos 2–4 dpf)

OR

(for embryos 5–16 dpf)

Reagent Prep

• Resuspend the collagenase in PBS 1X (ASW?) 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. #TODO: What is ceph equivalent of DMEM-10%FBS?
• Prepare the Dissociation Mix and keep at 30 °C. Each tube requires 500 μl of Dissociation Mix.

Protocol

Speciment Prep

1. Collect embryos or larvae at the desired stage.
2. Euthanize embryos? and quickly proceed to the next step.
3. CRITICAL: Do not freeze samples unless you are making nuclei. Freezing will destroy the cell membrane and make viable dissociation impossible.
4. Transfer embryos or larvae into 1.5 ml microcentrifuge tube
   • ∼ 30 embryos/tube for 2–4 dpf (Adjust accordingly for ceph)
   • ∼ 20 embryos/tube 4–10 dpf
   • ∼ 5 embryos/tube 10–16 dpf
5. Wash 2X in 1 ml ASW 1X

Enzymatic Dissociation

Dissociation with trypsin and collagenase

1. Remove ASW
2. Add 500 μl of Dissociation Mix (pre-heated at 30 °C) to each 1.5 ml microcentrifuge tube
3. 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.
4. Add 800 μl of DMEM-10%FBS to stop the dissociation.
5. 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

1. Discard the supernatant
2. Resuspend the pellet in 1 ml PBS 1X
3. Centrifuge 5 min at 700 g at RT
4. Filter single cell suspension
5. 1.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.
6. Filter through 70 μm nylon mesh into 50 ml conical tube
7. 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.

Alternative mechanical dissociation

• Use Ca2+ and Mg2+ free ASW to wash embryos (+ BSA)
• Gentle but thorough
• Can try TryplE
  • Sometimes they explode
  • May need salt supplement
    • 1:10 dilution of 5M NaCl
  • May or may not need to quench
• Can try papain
• 18ºC is ideal (no higher than 20ºC)

1. Input should be ~30-40 embryos at ~Stage 23

After dounce

1. Wash through 70 μm nylon mesh
2. Wash through 40 μm nylon mesh