Lab
Protocols
INSTITUTE OF MEDICAL BIOLOGY
Neural Stem Cell
Protocols
Neural Stem Cell Protocols
8A Biomedical Grove
#06-06 Immunos
Singapore 138648
Tel: (65) 64070150 Fax: (65) 64642049
shRNA knockdown in NSCs using lentivirus
Transfection of packaging cells (HEK293T) (version for unpurified virus) using
Effectene from Qiagen
1. Seed 0.4 million packaging cells in 35mmdish coated with poly-l-lysine. Use
low passage packaging cells.
2. Next day, add required amount of shRNA and packaging mix in Eppendorf
tube. For each 35mm dish, add
shRNA (in pll3.7 plasmid)
pMDL
pREV
pVSVG
2ug
1.3ug
0.5ug
0.7ug
To this, add 100ul EC buffer and mix well.
3. To above mixture, add 3.2ul enhancer (yellow cap) and mix well. Stand for
5min, RT.
4. To above, add 10ul effectene (green cap), mix and stand for 15 mins. Add
600ul of spent medium (medium from 35mm seeded dish), mix and add
dropwise back to seeded cells. Swirl. No medium change is required.
5. Collect medium (containing viral particles) 48 hours and 72 hours posttransfection. Store at 4 deg.
Viral titration (version for unpurified virus)
1. Seed 10,000 293T cells per well in 96-well plate (100ul medium each) (seed 2
days after transfection)
2. Next day, change to medium containing 8ug/ml polybrene.
3. Add 0ul, 2ul, 5ul, 10ul, 20ul, 30ul of unpurified virus (duplicates each)
4. Observe 2-3 days post-tranduction. For good titre, almost 100% cells should
be transduced for volumes between 5-20ul.
Transfection of packaging cells (HEK293T) (version for purified virus) using
Effectene from Qiagen
1. Seed 4 million packaging cells in 10cm dish coated with poly-l-lysine. Require
4x10cm dish per shRNA plasmid for 3 plasmids and collection over 2 days.
2. Next day, add required amount of shRNA and packaging mix in Eppendorf
tube. Add all in proportion to volumes for 35mm dish stated above.
To this, add 600ul EC buffer and mix well.
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3. To above mixture, add 16ul enhancer (yellow cap) and mix well. Stand for
5min, RT.
4. To above, add 60ul effectene (green cap), mix and stand for 15 mins. Add
350ul of spent medium, mix and add dropwise back to seeded cells. Swirl. No
medium change is required.
5. Collect medium (containing viral particles) 48 hours and 72 hours posttransfection. Store at 4 deg.
Viral purification by ultracentrifugation
1. Soak ultracentrifuge buckets and screws in 70% ethanol (minimum 2hrs,
maximum O/N)
2. UV Beckman tubes for 1hr. Place close to the UV lamp.
3. Filter viral supernatant using 0.4uM filter and vacuum (1 filter for 1 shRNA).
Use cellulose acetate low protein binding membrane filter.
4. Aspirate excess ethanol in the buckets and screws and leave them inverted on
c-fold to dry.
5. Add 33ml of viral supernatant into each Beckman tube. (2 tubes per shRNA)
6. Place tube in bucket and screw, one bucket at a time while the others are still
inverted on c-fold. Screw tight till the number on the bucket aligns with the
number o n the screw.
7. Hang buckets on SW28 rotor in following order (1,4,2,5,3,6). Place rotor in
ultracentrifuge and rotate by hand to hear sound. Close ultracentrifuge lid and
set settings (19600rpm, 22deg, 2hrs but for time put “on hold” and not 2 hrs in
case we are not available 2 hrs later).
8. Press “recall”, “enter” and “start”. Speed will go up till 3000rpm and vacuum
will blink at 75u. 5 mins later speed will reach 19600rpm and vacuum to 20u.
9. After 2 hrs, take out tubes immediately. Remove caps slowly and pour out
supernatant fast and invert Beckman tubes on c-fold.
10. Aspirate excess liquid on wall of tube. Add 300ul HBSS solution to resuspend
virus by pipetting up and down and use same 300ul HBSS to resuspend next
tube of the same shRNA. Transfer virus to Eppendorf tube. Aliquot virus in
25ul and 3ul volumes (eg: 10x25ul and 16x3ul).
11. Titre virus by FACS
Use double gloves, mask and follow proper disposal of viral materials.
Viral titration using FACS (version for purified virus)
Day 0
1. Coat the wells of a 24-well plate with poly-l-lysine.
2. Prepare 293T cell suspension of 80000 cells/ml and add 500ul of suspension
into each well. Incubate for 1 day.
Day 1
1. Aliquot 20ml of medium into a 50ml Falcon tube.
2. Add 20ul of 8mg/ml polybrene (PB) into the medium and mix well.
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3. Add 495ul of medium (with PB) into an eppendorf tube and 450ul of medium
(with PB) into each of 7 eppendorf tubes.
4. Thaw an aliquot of purified virus stock in ice.
5. Add 5ul of virus into tube with 495ul medium (100x dilution). Mix well by
pipetting and avoid bubbles.
6. Transfer 50ul of diluted virus into tube with 450ul medium (1000x dilution). Mix
well by pipetting and avoid bubbles.
7. Create serial diluted virus by transferring 50ul of suspension from newly mixed
tube into a tube with 450ul medium.
8. Aspirate medium from 24-well plate prepared on Day 0.
9. Add 400ul medium (with PB) into each well gently.
10. For each dilution of virus, add 100ul into each well in triplicates. Need
untransduced wells also.
11. Swirl plate and incubate for 3 days.
Day 4
1. Aspirate medium from 24-well plate.
2. Gently add 500ul of PBS into each well and swirl gently to wash. Aspirate out
PBS.
3. Add 200ul trypsin to each well and incubate at 37deg for 5 minutes.
4. Prepare 2% FBS by diluting with PBS and add 800ul of 2% FBS into each well
to inactivate trypsin.
5. Transfer contents into eppendorf tube and spin at 500g for 5 mins.
6. Aspirate supernatant gently and resuspend cell pellet in 300ul of 2% FBS and
transfer to a 5ml polystyrene round-bottom tube (with cell-strainer cap) (BD
falcon 352235).
Viral transduction of NSCs
1. Seed dissociated NSCs (20000 cells/ml) in 2ml NSC growth medium per well
in a 6-well plate.
2. Thaw purified virus on ice. Add virus at MOI=10 (add 400000 TUs per well).
3. Any concentration of polybrene is toxic to NSCs, so do not add polybrene to
medium.
4. Check for transduction 48 hrs later.
5. Can increase cell density of up to 50000 cells/ml.
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