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All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.
1. Manual Sorting of Fluorescently Labeled Mouse Neurons
- Pull glass microcapillaries (see Table of Materials) to 10–15 µm exit diameter using a capillary puller with the following settings: heat: 508, pull: blank, vel: blank, time: blank.
- Attach 120–150 cm flexible silicone tubing (~0.8 mm inside diameter) to a 0.2 µm polyvinylidene difluoride (PVDF) membrane syringe filter and a two-way tubing valve using suitable tubing connectors.
- Prepare 500 mL of chilled (4 °C) artificial cerebrospinal fluid (ACSF, Table 1), and oxygenate by bubbling 5% carbon dioxide balanced oxygen through an airstone for 15 min or until the solution clears completely.
- Prepare 100 mL of ACSF with a cocktail of activity blockers in a 150 mL beaker (Table 1).
- Prepare 100 mL of ACSF with 1 mg/mL Streptococcus fraction IV protease in a 150 mL beaker (Table 1).
- Prepare 100 mL of ACSF with 1% fetal bovine serum (FBS) solution in a 150 mL beaker and oxygenate with 5% carbon dioxide balanced oxygen through an airstone.
- Dissect the brain from an euthanized mouse by opening the cranium with a small scissor and extracting the fresh brain using fine forceps without damaging the cortex.
NOTE: Mice of any strain, age, and gender can be used as desired. Do not freeze the brain or perform manual sorting on mice injected with viruses or other hazardous/infectious agents.
- Keep the brain in chilled and oxygenated ACSF, leaving an airstone attached to 5% carbon dioxide balance oxygen during the entire duration of the sectioning.
- Position the brain on the vibratome chuck and cut coronal or sagittal sections at 300 µm thickness. Collect as many sections as needed to obtain a minimum of ~10–50 labeled cells up to several hundred cells. Put slices on a cotton meshed slice holder, placed inside a beaker so they are bathed in oxygenated ACSF.
- Move fresh slices into a beaker containing ACSF with activity blockers and block for 15–20 min at room temperature. Keep bubbling oxygen using airstone.
- Move the slices to a beaker containing ACSF with the protease solution to perform mild digestion at room temperature: 20–30 min for P4-14 animals or up to 45–60 min for P28-56 animals. Keep bubbling oxygen.
- Wash out ACSF with protease by moving slices back to the beaker containing ACSF with activity blockers solution for 5–10 min at room temperature. Keep bubbling oxygen.
- Move individual sections to a 100 mm Petri dish containing ACSF with 1% FBS at room temperature. Under a fluorescent dissection scope, microdissect areas and layers of interest having a minimum of 10–50 cells (up to a few hundred). Perform the microdissection with a pair of fine forceps or by attaching 22–28 G injection needles onto wooden holders (skewers).
- Using a Pasteur pipette, move the microdissected pieces to a 2 mL microfuge tube containing ~0.8 mL of 1% FBS in ACSF solution.
- Triturate the dissected tissue in the microfuge tube at room temperature. Make three long-stemmed Pasteur pipettes with decreasing exit diameters by rolling them over an open flame. Perform ~10 strokes with each pipette, starting with the biggest and ending with the smallest.
- Dispense the dissociated cells into a 100 mm Petri dish containing oxygenated ACSF (Petri dish can be thinly coated with 5 mm of 1% agar or clear silicone compound at 1:10 ratio, if desired). Keep at room temperature.
- Wait ~5-7 min for the cells to gradually settle, then observe the GFP/RFP signal under a dissection microscope.
NOTE: Single cells, debris, and small clumps will be visible in bright-field (BF).
- Pick 10–15 GFP/RFP cells at a time using a capillary pipette (from step 1.1) attached to flexible silicone tubing (0.4–0.8 mm inner diameter) and dispense the cells into a 100 mm Petri dish containing fresh oxygenated ACSF.
- By blocking the end of the tubing valve with the tongue, position the capillary close to the cell of interest. Capture cells using capillary action upon relieving the block, and quickly block again to prevent excess fluid from entering the pipette. Dispense the cells onto a 100 mm Petri dish with fresh oxygenated ACSF by gently blowing, while observing the capillary tip under fluorescence optics of the dissection microscope. Aim to collect ~100–150 cells total.
- Repeat step 1.19 once or twice more (depending on debris) and transfer a total of ~50–75 cells to a new 100 mm Petri dish, making sure that contaminants such as debris are minimal in bright-field differential interference contrast (DIC) optics.
Table 1: List of solutions and buffers.
| Buffer | Item | Concentration | Amount (µL) |
| Sample Collection buffer | Recombinant ribonuclease inhibitor | | 55 |
| RCC | 1:50K diluted | 110 |
| Nuclease free water | | 605 |
| Aliquot 43.75 µL of above into 16 tubes (2 strips of 8; 200 µL PCR tubes); add following per tube |
| UMI-primers (e.g. N10B1-N10B16) | 1 ng/µL | 6.25 µL/tube |
| Final volume in each tube 50 µL (each tube can be split in 25 µL aliquots and frozen at -80 °C) |
| Solutions: | Item | Concentration | Amount |
| CSF: to make 5 L dissolve | NaCl | 126 mM | 36.8 g |
| KCl | 3 mM | 1.15 g |
| NaH2PO4 | 1.25 mM | 0.75 g |
| NaHCO3 | 20 mM | 8.4 g |
| To 500 mL of ACSF, bubble oxygen for 10-15 min then add following fresh each time: |
| D-glucose | 20 mM | 1.8 g |
| MgSO4 | 2 mM | 0.5 mL from 4 M stock |
| CaCl2 | 2 mM | 0.5 mL from 4 M stock |
| Keep ACSF oxygenated through out |
| Solutions: | Item | Concentration |
| Activity blocker cocktail: make a 100x stock |
| To 100 mL ACSF add | | |
| APV | 0.05 mM | |
| CNQX | 0.02 mM | |
| TTX | 0.0001 mM (0.1 µM) |
| Solutions: | Item | Amount | |
| Protease soltion (100 mL) | Protease from Streptomyces griseus | 100 mg | |
| Fetal bovine serum: commercial source, aliquot in 500 µL for each use. |