Brain Cell Isolation from Zebrafish Larvae: A Technique to Obtain Intact Neurons, Macrophages, and Microglia from Zebrafish Brain Tissue

All procedures involving human participants have been performed in compliance with the institutional, national and international guidelines for human welfare and have been reviewed by the local institutional review board.

1. Homogenization

NOTE: All steps are performed 4 °C.

1. Add 1.5 mL Tricaine (15 mM) to 90 mm Petri dishes containing 50 larvae in 50 mL of E3 embryo medium treated with 200 µM PTU to terminally anesthetize them.
   1. Suck up 10 anesthetized larvae from the Petri dishes with a 3 mL Pasteur plastic bulk pipette.
   2. Transfer anesthetized larvae 10 by 10 into a 55-mm Petri dish filled with ice-cold E3 embryo medium + Tricaine.
   3. Under a stereomicroscope, align 10 larvae in the center of the Petri dish. Then, transect larval heads above the yolk-sac using surgical micro-scissors (exclude swim bladder to avoid floating heads).
   4. Suck up heads from the Petri dishes with a 3 mL Pasteur plastic bulk pipette. Wait until all heads gather within the pipette tip and then transfer them into a glass homogenizer containing 1 mL ice-cold Media A (transfer in a minimal volume to reduce Media A dilution by E3 + Tricaine). Keep the glass homogenizer on ice. Use one homogenizer per experimental condition.
   5. Replace each small Petri dish containing ice-cold E3 + Tricaine with a new one every 30 min to assure that transection is performed in cold E3 + Tricaine medium.
   6. Replace the ice-cold Media A in the glass homogenizer when the color starts fading.
      NOTE: Media A dilution can alter the head tissue due to temperature changes.
   7. Once all heads have been collected (600 heads/condition), remove the maximum volume of Media A from the glass homogenizer and replace it with 1 mL of fresh ice-cold Media A.
   8. Disrupt the brain tissue with a tight glass homogenizer on ice. Perform 40 rounds of crushing and turns for 3–5 dpf larvae and 50 for 7 and 8 dpf larvae.
2. Add 2 mL of Media A to cell suspension (1 mL Media A/200 heads), which will dilute cells and reduce their agglomeration with myelin to facilitate their separation during the centrifugation in density gradient medium.
   1. To eliminate cell agglomeration, run the cell suspension through a 40 µm cell strainer placed on top of a cold 50 mL falcon tube maintained on ice. Repeat this operation 3 times.
   2. Transfer 1 mL of cell suspension into cold 1.5 mL tubes and spin them at 300 g for 10 min at 4 °C.
   3. Remove supernatant using a 10mL syringe + needle 23G x 1''.
3. Resuspend the cell pellet with 1 mL of ice-cold 22% density gradient medium gently overlaid by 0.5 mL of ice-cold DPBS 1x (do not mix them, an interphase between both solutions will be seen).
   1. Spin tubes at 950 g without brake and slow acceleration for 30 min at 4 °C.
      NOTE: This step separates myelin from other cells by sequestering it at the interphase of DPBS 1x and 22% density gradient medium, whereas cells will pellet at the bottom of the tube. Myelin removal is more efficient when cell concentration is not too high.
   2. Using a 10 mL syringe + needle 23G x 1'' discard the maximum of DPBS, density gradient medium, and myelin trapped at their interphase.
   3. Wash cells with 0.5 mL of Media A + 2% of normal goat serum (NGS), then spin tubes at 300 g for 10 min at 4 °C.
   4. Discard the maximum of the supernatant, then pool all cell pellets from the same experimental condition together in 1 mL of Media A + 2% NGS.
4. If the cells of interest express a fluorescent protein-like macrophages/microglia from mpeg1:eGFP or neurons from NBT:DsRed transgenic fish, run the cell suspension through a 35 µm cell strainer cap and transfer them into a cold 5 mL FACS tubes on ice, protected from light.
   NOTE: Alternatively, immunostaining of microglia can be performed.