TITLE: Human Spinal Cord Nuclei Isolation
AUTHORS: Abbas Rizvi, Elena Kandror, Tom Maniatis

[STEPS]
?. [Buffer Prep]
10x Stock Salt Solution: (raise to 50mL in water)note: can be stored at  for up to 2 weeks                                                                          Final Concentration CaCl2 (1M)                                         50mM Magnesium Acetate (1M)                30mM Tris pH7.8 (1M)                                     100mM 2-Mercaptoethanol (14.3M)               10mM
4 °C
2.5 ml
1.5 ml
5 ml
35 µl
?. [Homogenization]
Critical Note: Perform all steps on ice! Add  1x Homogenization Buffer into a clean 7ml capacity dounce homogenizernote: if using NeuN antibody, add it here (1:500 final dilution = 20ul primary labeled NeuN antibody)
0 °C
4 ml
?. [Buffer Prep]
50% Optiprep: (raise to 50mL in water)note: can be stored at  for up to 2 weeks Optiprep 10x Stock Salt Solution Sucrose (2M)
4 °C
25 ml
5 ml
8 ml
?. [Buffer Prep]
29% Optiprep: (raise to 50mL in water)note: can be stored at  for up to 2 weeks Optiprep 10x Stock Salt Solution Sucrose (2M)
4 °C
14.5 ml
5 ml
8 ml
?. [Buffer Prep]
1x Homogenization Buffer: (raise to 10mL in water)note: prepare fresh each time, keep on ice                                                                              Final Concentration 10x Stock Salt Solution                           1X Sucrose (2M)                                         320mM EDTA (5mM)                                          0.1mM NP-40 (10%)                                           0.1% PMSF (100mM)                                       100uM DAPI (5mg/ml) -- optional
1 ml
1.6 ml
200 µl
100 µl
10 µl
5 µl
?. [Homogenization]
Cut 4 frozen tissue slices ~0.2mm thick and transfer into dounce homogenizer.
?. [Homogenization]
Homogenize tissue with:15 strokes loose pestle20 strokes tight pestle(avoid bubbles)
?. [Homogenization]
Filter the sample through a 100um mesh into a fresh 15ml tube; rinse the filter with  1x Homogenization Buffer and collect in the same tube.
1 ml
?. [Homogenization]
Incubate
?. [Homogenization]
Add equal volume 50% Optiprep (~)
4.5 ml
?. [Homogenization]
Invert thoroughly but gently to mix (avoid bubbles). This becomes a 25% optiprep solution.
?. [Homogenization]
Gently layer over  29% Optiprep in swinging bucket ultracentrifuge compatible tube (we use Beckman #344058)
10 ml
?. [Homogenization]
Spin in a swinging bucket centrifuge at 10,100g (=7400rpm in SW-28 Beckman rotor) for   at note: acceleration and break both on at max (setting 9)Once the spin is done, the nuclei will be pelleted at the bottom of the ultracentrifuge tube. However, don't disrupt the gradient as doing so can introduce myelin contaminant into the sample!
4 °C
?. [Nuclei Resuspension]
Going from the top of the solution, remove the 25% optiprep layer using a 1ml pipet.note: if there is myelin coating over the top of the solution, remove it first.
?. [Nuclei Resuspension]
Once the 25% optiprep layer is mostly removed, continue to pipet out solution from the top of the tube. The most effective way to do this while preventing myelin carry-over is to "slurp" the solution with a 1ml pipet such that you are effectively aspirating bubbles (50% solution and 50% air).
?. [Nuclei Resuspension]
Once there is ~5ml solution left, pipet the solution out normally, 1ml at a time. Do not reuse the pipet tip. Remove the entire volume, being very careful with the last aspiration so as not to disturb the nuclei.
?. [Nuclei Resuspension]
Resuspend the nuclei in your buffer of choice (eg, 1mL of PBS + 0.1% BSA) by forcefully expelling the buffer directly against the bottom of the ultracentrifuge tube. Pipet up and down to fully resuspend the nuclei, and transfer to an ice-cold 1.5mL tube.