1. Timed mating was set up by placing one male and two female C 57BL/6 mice
2. The female was then checked for a vaginal plug early the following morning. 3. Embryonic ages were determined as the time since the appearance of the
copulative plug (the first day was determined as E0.5).
4. Pregnant female at the specified gestational age of 14.5 days (E14.5) were killed
5. The uteri were aseptically removed and transferred to a Petri dish containing
sterile Dulbecco’s phosphate-buffered saline (Invitrogen) with 30% glucose
(Sigma) and 2% penicillin/streptomycin (Invitrogen).
6. Fetuses were removed from the amniotic sac and transferred to a new Petri dish
containing ice-cold Hanks’ balanced salt solution.
7. Cortices were rapidly excised from the fetuses.
8. Cortices were mechanically dissociated by pipetting into a single cell suspension. 9. Cells were plated at a density of 2 x 105 cells/ml into 10 cm culture dishes (NUNC)
in Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 (1:1) mixture
medium (Invitrogen) containing B27 supplement (Invitrogen), 20 ng/ml bFGF
(Sigma), 20 ng/ml EGF (Invitrogen) and 1% penicillin/streptomycin (Invitrogen).
Passaging of NSCs/NPs
1. Floating neurospheres with diameter were passaged every 4-6 days.
a. Solution 1: 9.5 ml media + 0.5 ml 1 N NaOH
b. Solution 2: 9.5 ml media + 0.5 ml 1 N HCl
3. The neurospheres and medium were transferred from the culture dish to a 15 ml
4. Centrifuged for 1 minute at 1000 rpm.
5. Remove the supernatant completely. 6. Resuspend the pellet in 600 ul medium. Pipette gently to resuspend
7. Aliquot the cells into 3 eppendorf tubes, 200 ul each. 8. Add equal volume of Solution 1.
9. Mix cells gently by pipetting up and down 3 times using a P200 tip. 10. Set the timer for 7 minutes.
11. After 2 minutes, mix cells gently by pipetting up and down 7 times. 12. After 3 minutes, mix cells gently by pipetting up and down 7 times. 13. After 7 minutes, mix cells gently by pipetting up and down 9 times.
14. Add the dissociated cells (400 ul) to 2 ml of culture medium containing equal
15. The cells from neurospheres are now in a single cell suspension which is ready
Storage of NSCs/NPs
1. Pool the culture medium with the neurospheres from the culture dish into a 50 ml
2. Centrifuge for 2 minutes at 1000 rpm.
4. Re-suspend the cells in 10.0 ml culture medium containing 10% DMSO.
5. Gently mix the neurospheres in the medium.
6. Aliquot 1 ml of the mixture into polypropylene cryovials.
7. Place the tubes in Mr. Frosty Freezing Container (NALGENE).
8. Transfer the container to –80°C freezer overnight.
9. The following morning, transfer the vials to a liquid nitrogen cryofreezer.
FACS sorting of NSCs/NPs
1. Dissociate the neurospheres into a single-cell suspension.
2. Wash the cells in PBS buffer to remove any residual growth factors that may be
3. Block the cells with 3% BSA in PBS buffer for 15 minutes at room temperature
4. Transfer 25 ul of the BSA-blocked cells (1 x 105 cells) to a 5 ml tube.
5. Add 10 ul of fluorescein-conjugated antibody.
7. Following this incubation, remove unreacted antibody by washing the cells twice
8. Finally, resuspend the cells in 200 – 400 ul of PBS buffer for FACS.
9. As a control, cells in a separate tube should be treated with isotype control.
96 well clonal nsph growth
1. Dissociate the neurospheres into a single-cell suspension.
2. Filter the suspension through a 45-um sieve
3. Collect the cells via centrifugation at 1000 rpm for 5 minutes.
5. Set the gating parameters with forward and side scatter to exclude debris, and
6. Prepare 96-well plate containing 200 ul of growth medium in each well.
8. On day-7, remove 100 ul of medium and add 100 ul of fresh medium into each
ScPCR Manual RT reaction
1. Thaw reagents (except enzymes). Get some ice.
2. Prepare Mix A (following is for 1 well, 7.25ul per well):
3. Add 7.2ul of Mix A into each well of 96-well place, using multichannel pipet.
5. Heat plate in PCR machine: Program #RT70 under HUIMIN
6. Chill the plate on ice and spin down.
7. Prepare Mix B while spinning plate (following is for 1 well, 2ul per well)
8. Add 2ul of Mix B into each well of 96-well plate.
10. Heat plate in PCR machine: Program #MMLV_RT under HUIMIN
11. Proceed with PCR or keep in -20 0C for use within a week.
PCR Reaction
1. Pipet 5ul of RT reaction into a new 96-well plate.
2. Prepare MasterMix (30ul reaction): (following is for per tube)
3. Add 25ul of MasterMix into each tube using multichannel pipet.
5. Warm up PCR machine. Use Program #SCRTPCR under HUIMIN.
6. Centrifuge the plate at 1200rpm, 25ºC for 2 min.
7. Load plate into PCR machine and start.
8. After PCR is complete, keep in -20ºC or proceed with 2nd round (Nested)
1. Prepare MasterMix (26ul reaction): (following is for per tube)
2. Pipet 1ul of sample from Round 1 PCR into a new plate
3. Add 25ul of MasterMix to each tube. Cap plate tightly.
4. Warm up PCR machine. Use program: Program #SCRTPCR under HUIMIN.
5. Centrifuge plate at 1200 rpm, 25ºC for 2 min.
7. After PCR is complete, keep in 4ºC fridge for use within a week (or 20 ºC for
longer storage) or proceed with DNA agarose gel.
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INSTRUCTIVO PARA LA VINCULACIÓN CON TERCEROS 1.- OBJETO El presente instructivo brinda información clara y sencil a para la vinculación de la UNLP conterceros, con la finalidad de cumplimentar los requisitos legales que regulan la materia a losefectos de salvaguardar las responsabilidades que, en materia civil, económica y/o penal, sepueden derivar de los compromisos asumidos. Asimismo,