1. Dissection

   can do on counter (i.e., in non-sterile conditions)
   use P8 rat pups, or P5 mouse pups
   keep on ice whenever possible
   for each step, do all pups, before moving to next step
   
   fill 2 tubes with approx. 30ml HHGN (one for dissection; other for cell processing -- keep sterile); keep on ice
   fill ice tray
   sterilize tools by soaking in EtOH: large forceps, 2 fine forceps, curved forceps, 1 small and 1 large scissors
   place TDn, DnB at room temp to thaw
   

   1. isolate cerebella (ÒCbÓ)

      cut off head into plate with HHGN
      hold nose with large forceps
      cut with scissors thru skin and skull, from side of neck, to top of head, across, and back to neck
      remove flap of skin/skull from front to back
      pinch off cerebellum with fine forceps, into HHGN
      

   2. remove meninges

      under dissecting scope, pull away using two fine forceps
      not necessary to remove entirely
      

   3. chop each cerebellum into approx 4 pieces ( not necessary for mouse, if Cb small)

2. Processing

   in tissue culture hood; keep sterile
   
   transfer tissue to 15ml pop-top tube (use 25 ml pipet so opening is large)
   rinse 3 X 2ml HHGN
   digest 15min, room temp, in 5 ml TDn (lower volume OK if doing individual Cb, eg. for mice)
   remove sup, wash 3 X 3ml HHGN
   add 5 ml DnB; transfer to 50 ml tube
   triturate with 5 ml pipet until homogeneous (20 X)
   let settle 5 minutes to remove clumps; transfer sup to second tube
   
   (optional:)
   re-triturate clumps with 5ml more DnB (more vigorously; seal pipet tip, triturate approx. 10X)
   let settle 5 minutes; transfer/pool sup with first sup
   
   centr 500 RPM, 3min, R.T.
   resusp pellet in 10ml culture media (should resuspend easily)
   count live/dead cells: eg.:
   dilute 1/10: 65ul media (or HHGN) + 25ul 0.4% trypan blue + 10ul cells [or, dilute 3/4 if low conc cells]; count 10ul
   expect approx 50% live cells
   
   dilute cells in media, and seed plates/wells:
   24well: 5 X 105 live cells 0.5 ml 35 mm: 2.5 X 106 1.5 ml 60mm: 5 X 106 3 ml 100mm: 1.5 X 107 9 ml note, 24w: shake plates vigorously left-right, top-bottom, before placing into incubator, to evenly distribute cells donÕt need to change media after seed (eg., 2h) grow in 5% CO2, at 37 C on 1 DIV (approx 24h), add araC to 10 uM: 24 well: 10ul 500uM 35mm: 30 ul 500uM [araC: -80C box G5 (HD) or E1] 60mm: 60ul 500uM feed with new media (containing araC) on (3)-4 DIV: 1ml/3ml per 60mm 200ul/500ul per 24 well [can do when replace media after transfection]

3. Death Induction

   on 6 DIV (have also done on 7 DIV) wash 2X with -KI 24w: 0.5 ml 35mm: 1.5ml 60mm: 2ml add -KI or +KI

1. HHGN

   500ml 1X HBSS 50ml 10X [without Ca, Mg; Gibco/BRL # 14180-020] 2.5mM Hepes pH 7.3-7.5 1.25 ml 1M glucose 7ml 2.5M (45%) NaHCO3 2ml 1M store at 4C (approx 2 mo.)

2. DNAase Boehr-Mann #104159 (2,000u/mg); bovine pancrease, grade II stock: 4,000 u/ml in H2O; sterile filter, store at -80C

3. TDn 250 ul DNAase 5ml HHGN 50mg trypsin [Worthington # 3703] pH with 0.1N NaOH sterile filter note, can make up large amount, aliquot to 5 mls, and store at -80C (approx 1 mo.) thaw to RT before use

4. DnB 500ul DNase I 10 ml BME [Sigma #B1522] can make up large amount, aligout to 10 mls, and store at -80C thaw to RT before use

4. 3 M KCl may need to autoclave to sterilize; too viscous to filter ?

5. BME+K 500ml BME [5mM K] 4.1 ml 3 M KCl (therefore, 24mM additional, 29mM K total) [unopened BME bottle: 523ml on average]

6. Calf Serum [Hyclone# A2151] need to heat inactivate sterile filter; aliquot to 50ml and 10.5 ml; store at -20C

7. Culture Media (ÒCbC MedÓ) 100 ml BME+K 10 ml calf serum 1 ml 0.2 M glutamine [use fresh tube] 1 ml Penn-Strep when refeed, add araC to 10uM: 40ul 25mM per 100ml final [K]= 26mM

5. -KI 100 ml BME [5mM K] 1 ml 0.2 M glutamine 1 ml Penn-Strep 40 ul 25 mM araC [10 uM final]

6. +KI per 10ml: 10 ml -KI 25 ul 2mg/ml insulin [5ug/ml final] 67 ul 3M KCl [20mM additional, final = 25mM] sterile filter (if insulin stock is unsterile)

usually use P8 Long-Evans rats; have also used P5-9 successfully; P5/6 rats yielded considerably fewer cells, and a relatively higher number of glia; published protocols also use Sprague-Dawley and Wistar rats yield of cells is approx 1.5 X 107 cells per P8 rat original protocols call for fetal bovine serum; however, calf serum was used and seems satisfactory insulin is used for survival, rather than IGF-1 of ref 1, due to cost although ref 1 reports survival in KCl or IGF (in place of serum, at 6DIV), mouse Cb cells survive better with both present (for rat Cb cells, KCl alone allowed full survival) without media change, in CbC media (serum + KCl), rat Cb cells live only about 7-9 days; with 1 change (4DIV), live approx 12 d

1. DÕMello et al. (1993) PNAS 90, p. 10989

2. Galli et al. (1995) J. Neurosci. 15, p. 1172