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Single-embryo single-cell dissociation protocol.md

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Single-embryo single-cell dissociation protocol

To obtain high-quality data, we developed and optimized a single-embryo single-cell dissociation protocol that varies based on the 10 developmental stages we studied. We used a very gentle dissociation method for early stages, and a harsher method for later stages. Our protocol utilized a combination of chemical and mechanical methods to achieve the highest possible dissociation efficiency while also maximizing cell viability.

All steps should be performed under sterile conditions to prevent contamination.

Materials:

  • Zebrafish embryos
  • 1mg/ml Pronase solution
  • PBS + 2% BSA
  • FACSmax solution (Gentalis - T200100)
  • 0.25% Trypsin-collagenase (100 mg/ml) solution
  • 1X PBS + 1% BSA solution
  • 20 μm mesh
  • 1X PBS+ 0.1% BSA + 18% optiprep solution
  • Cellometer auto T4 cell counter (Nexcelom biosciences)
  • Trypan blue solution

Procedure:

  1. Dechorionate the zebrafish embryos using 1mg/ml Pronase solution.
  2. Precoat 1.5 ml Eppendorfs with PBS + 2% BSA at room temperature for 15 minutes.
  3. For embryos between 10 hpf to 1 dpf, dissociate a single embryo in 50 µl of FACSmax solution. For embryos between 2 dpf to 10 dpf, dissociate a single embryo in 50 µl of 0.25% Trypsin-collagenase solution.
  4. Pipette and flick the Eppendorf gently to dissociate the embryo without forming bubbles.
  5. Centrifuge the eppendorf by gradually increasing the speed from 300 rcf for 2 mins(early stages) to 750 rcf for 7 mins (later stages) at 4°C depending on the stage of interest.
  6. Carefully remove the dissociation media without dislocating the pellet. Wash the pellet with 1X PBS + 1% BSA solution.
  7. Pass the solution through a 20 μm mesh for later stages (2dpf – 10 dpf) to remove undissociated debris. Centrifuge the solution again at 4°C with the same conditions. Discard the supernatant and dissolve the pellet in 25 µl of 1X PBS+ 0.1% BSA + 18% optiprep.
  8. Measure the cell concentration using the cellometer auto T4 cell counter. Dilute the cell suspension and trypan blue solution (1:4) to measure the cell count.
  9. Repeat the above steps for each of the required stages, using a single embryo per experiment and 4 replicate experiments per stage (4 siblings).