Materials Two adult mice HibernateA * ( A for adult) Papain B27 minus retinyl acetate * Glutamax or L-Gln OptiPrep density 1.32 (Sigma): 60% (w/v) solution of iodixanol ( 碘克沙醇） in water (sterile) * NeurobasalA* ( A for adult) Growth factors: bFGF, EGF, PDGFbb* P/S (Penicillin streptomycin) or Gentamycin Surgical equipment 15 ml and 50 ml centrifuge tubes (new, better to be corning, NEST or BD). Pasteur pipette (or common dropper) 0.22 m filter 40 m cell strainer Swinging bucket centrifuge Ultralow adhesion plastic culture dishes ( or common dishes made in China, but not corning treated dishes) * New plastic pipette tips Trypan blue Hemacytometer
Reagent and equipment setup HABG (40ml-60ml): HA, B27 minus retinyl acetate, 0.5mM Glutamax Culture medium: NeurobasalA, B27 minus retinyl acetate, 0.5mM Glutamax, P/S, bFGF (10ng/ml), EGF (10ng/ml), PDGFbb(10ng/ml), heparin (2 g/ml, optional). Papain: >34U/ml stock (final concentration 34U/ml), 37 ℃ for 20–30 min, filter- sterilize into tubes. Disinfect surgical equipment with 70% ethanol.
Step 1. Tissue dissection Anesthetize adult mice. Disinfect head with 70% ethanol, and expose the brain. Transfer the brain to a dissection dish with HibernateA (or PBS) at 4 ℃. Dissect hippocampi into HABG at 4 ℃.
Step 2. Digestion Cut hippocampi in HABG into small pieces (1mm 3 ) (the smaller the better). Add papain to a final concentration of 34U/ml. Incubate at 30 ℃ or 37 ℃ for 30 min (better to shake). Transfer the tissue to a 15- ml tube and centrifuge at 200g for 3 min. Discard the supernatant. Re-suspend the tissue with 2 ml HABG.
Step 3*. Release Cells from Tissue ----determine your yield Trituration with pasteur pipette (or dropper) (*1- ml pipette tip is too sharp), without air bubbles for several times (determined by yourself). Allow the pieces to settle for 1 min and transfer the supernatant to an empty 15-ml tube. * Re-suspend the sediment from the first tube in 2 ml HABG, repeat the last two steps twice more. Finally, you got 6- ml suspended cells. Filter the suspended cells with cell strainer.
Step 4*. Density Gradient Centrifugation Prepare density gradient. * ( Be careful) Carefully apply the cell suspension to the top of the prepared OptiPrep density gradient. Cenfrifuge the gradient at 800g (1,900 r.p.m. in a swinging bucket centrifuge) for 15 min at 22 ℃ (or at room tempreture).
Collect fraction 3 into a new 15- ml tube. Wash out the gradient material with 4- to 5- ml HABG, centrifuge at 200g for 2 min, and discard the supernatant. Repeat washing. Re-suspend the cell pallet with 0.5- to 1- ml culture medium.
Step 5. Cell Plating Aliquot 10 l of cell suspension into a small tube containing 10 l trypan blue. Mix and apply approximately 10 l to a hemacytometer. Count phase bright spherical cells. Plate cells at a low density of 4000 to 8000 cells/ ml for clones of neurospheres in ultralow adhesion substrate. Culture at 37 ℃, in a 5% CO2, 9% O2 gas (optional), humidified incubator.
Critical factors Optimized protease digestion Control of osmolarity and pH outside the incubator Density gradient separation Low-adhesion plastic substrate B27 minus retinyl acetate Suitable growth factors 9%O 2, 5% CO 2.