Gaspard protocol by nuhman10

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									Neural differentiation protocol with DDM
Nicolas Gaspard et al. 08/2008 / Pierre Vanderhaeghen lab
See also Nature 455 (2008), 351-357.



Notes :
   -   Different ES cell lines grow at different rates in ES medium but also in DDM ;
       one should adjust the seeding density before differentiation at day-1 for each
       cell line. The densities suggested below work well for E14Tg2a and Tau-GFP
       cells.
   -   The “classical protocol” consists of a first period of differentiation on gelatin-
       coated plastic dishes then a transfer on polylysin/laminin-coated glass
       coverslips for easy immunofluorescence. Alternatively, cells may be seeded
       on gelatin-coated coverslips already on day -1. Seeding density should be
       lowered ( 0,3-3x103 cells/cm2 works well for E14Tg2a cells).



Day -1 :
   -   Prepare gelatin-coated dishes

       For feeder-free ES cell lines

   -   Rinse ESC with pre-warmed PBS
   -   Add pre-warmed trypsin/EDTA (1ml/5cm dish or 2,5ml/10cm dish) and wait for
       cells to detach from the plate.
   -   Inhibit trypsin with pre-warmed ES medium (4ml/5cm dish or 10ml/10cm dish)
       and recover all the cells by gently flushing the bottom of the plate
   -   Centrifuge at 1200rpm for 3 minutes
   -   Remove supernatant and resuspend ESC in ES medium
   -   Dissociate thoroughly using a Pasteur pipette
   -   Count the cells
   -   Seed ESC on gelatin-coated dishes at a density of 5-10x103 cells/cm2

       For ES cell lines growing on feeders

   -   Rinse ESC with pre-warmed PBS
   -   Add pre-warmed trypsin/EDTA (1ml/5cm dish or 2,5ml/10cm dish) and wait for
       cells to detach from the plate.
   -   Inhibit trypsin with pre-warmed ES medium (4ml/5cm dish or 10ml/10cm dish)
       and recover all the cells by gently flushing the bottom of the plate
   -   Centrifuge at 1200rpm for 3 minutes
   -   Remove supernatant and resuspend ESC in ES medium
   -   Dissociate thoroughly using a Pasteur pipette
   -   Plate all the cells (feeders+ESC) on a gelatin-coated 10cm Petri dish. Place
       the cells back in the incubator; feeders will start attaching before ESC. Check
       the dish under the microscope every 5 minutes until most of the big cells
       (feeders) have attached and only small cells can be seen floating. This usually
       takes between 15 and 30 minutes
   -   Carefully transfer the supernatant containing the ESC to a new Falcon tube.
   -   Count the cells
   -   Seed ESC on gelatin-coated dishes at a density of 5-10x103 cells/cm2



Day 0 :
   -   Remove ES medium
   -   Rinse once with pre-warmed PBS
   -   Add DDM (5ml/5cm dish or 10ml/10cm dish)


Day 2 to 10-14 :
   -   Change medium every other day
   -   Cyclopamine (1M) is added in the medium from day 2 to day 10


Day 10-14 :
   -   Prepare polylysin/laminin-coated coverslips

   -   Rinse cells once with PBS
   -   Add trypsin/EDTA and wait for cells to detach
   -   Inhibit trypsin with pre-warmed PBS with 10% serum (4ml/5cm dish or
       10ml/10cm dish) and recover all the cells by gently flushing the bottom of the
       plate
   -   Centrifuge at 1200rpm for 3minutes
   -   Remove supernatant and resuspend ESC in N2B27
   -   Dissociate thoroughly using a Pasteur pipette
   -   Count the cells
   -   Seed cells on polylysin/laminin-coated coverslips in 12-wells plates at a
       density of 100-400x103 cells/cm2


Day 10-14+1 to 21-28 :
   -   Change the medium every other day
ES medium

DMEM                                         ad 500ml
+    Fetal bovine serum                      50ml
+   Non Essential Amino Acids 100x           5ml          ( 1x)
+   Glutamine 200mM                          5ml          ( 2mM)
+   Sodium pyruvate                          1ml          ( 1 mM)
+   Penicillin/Streptomycin                  5ml
+   ß-Mercapto-Ethanol                       1.7µl        (0.055mM)


DDM (Defined Default Medium)

DMEM/F12 + Glutamax                           ad 500ml
+   N2     100x                              5 ml         ( 1x)
+   BSA V 7.5%                               3.33ml       (500 µg/ml)
+   ß-Mercapto-Ethanol                       3.5µl        (0.11mM)
+   Non Essential Amino Acids 100x           5ml          ( 1x)
+   Sodium pyruvate                          1ml          ( 1 mM)
+   Penicillin/Streptomycin                  5ml

All solutions from Gibco/Invitrogen except ß-Mercapto-Ethanol from Sigma-Aldrich.
Filter and store at +4°C.

Cyclopamine is from Calbiochem. Stock solution is at 1 mM in absolute ethanol.
Store at -20°C. Add just before warming medium.


N2B27

A 1:1 mixture of:

DMEM/F12                                     ad 500ml
+   N2     100x                              5 ml         ( 1x)
+   BSA V 7.5%                               3.33ml       (500 µg/ml)
+   ß-Mercapto-Ethanol                       3.5µl        (0.11mM)
+   Non Essential Amino Acids 100x           5ml          ( 1x)
+   Sodium pyruvate                          1ml          ( 1 mM)
+   Penicillin/Streptomycin                  5ml

and:

Neurobasal                                   ad 500ml
+    B27 (without vitamin A; 50x)            10ml ( 1x)
+    Glutamine 200mM                         5ml
+    Penicillin/Streptomycin                 5ml

All solutions from Gibco/Invitrogen except ß-Mercapto-Ethanol from Sigma-Aldrich.
Filter and store at +4°C for up to one week.
Trypsin/EDTA

Trypsin 0.05%
EDTA 0.5mM
in PBS


Gelatine coating

Coating solution is :

- Gelatin 0,1g/100ml
- in H2O Analar

Gelatin is from Sigma-Aldrich.
Autoclave and store at +4°C for up to one week.
Coat for one hour at room temperature then remove by aspiration. Let dry under the
hood.




Polylysin/Laminin coating

Stock solutions are :

- Polylysin 500 µg/ml in PBS
Polylysin is from Becton Dickinson. Store at -20°C
- Laminin 30 µg/ml in PBS
Laminin is from Becton Dickinson. Store at -80°C

Coating solution is a mixture of:

- Polylysin 33 µg/ml
- Laminin 3 µg/ml
- in PBS
Prepare fresh each time. Coat for 2 hours at +37°C. Rinse three times with PBS and
dry by aspiration.


Coverslip processing

- Wash coverslips in 1N HCl for at least 1h
- Rinse twice in Analar water
- Rinse twice in absolute ethanol
- Air dry under a hood
- Sterilize overnight in an oven at 180°C
Immunofluoerscence on glass coverslips

   -   Remove culture medium

   -   Fix the cells with 4% PFA (pH7,4) for 30min at 4°C

   -   Rinse three times with PBS at room temperature

   -   Incubate 30min at room temperature in blocking/permeabilization buffer (see
       below)

   -   Incubate overnight at 4°C with the primary antibody in antibody buffer (see
       below)

   -   Wash three times with PBS

   -   Incubate for 2hours at room temperature with the secondary antibody in
       antibody buffer supplemented with 5% Horse Serum

   -   Wash three times with PBS

   -   If needed, repeat the incubation and washing steps with another pair of primary
       and secondary antibodies

   -   Nuclear counterstaining by incubation for 5min in bisbenzimide solution(1g/ml
       in PBS)

   -   Rinse once with PBS

   -   Mount in suitable medium (we use Glycergel from DAKO)



Blocking/Permeabilization buffer :

   -   PBS

   -   BSA 1g/100ml

   -   Triton 0,3%



Antibody buffer :

   -   PBS

   -   BSA 1g/100ml

   -   Triton 0 ,1%

								
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