Preparations before day of culturing:
- Prepare sterile dissecting solution (DS).
- Prepare NBM/B27 (Neurobasal Mediom with B27 supplements).
- Autoclave ddH2O and sterilize glass coversilps, if needed.
- Coat tissue culture dishes or glass coverslips with poly-D-lysine.
Poly-D-Lysine Coating:
Prepare the day before culturing under sterile conditions.
- Thaw aliquot of 10X PDL and place on ice.
- Add 9 ml sterile ultra-filtered water to 1 ml of PDL and mix well (1X).
- Coat surfaces with 1X PDL overnight at room temperature as follows:
- glass coverslips (Bellco Glass, Inc. Cat.# 1943-00012): 75 µl/coverslip
OR
- 24 well culture plate: 300 µl/well
OR
- 35mm culture dish: 1ml/dish
- Rinse 5X with sterile water (use sterile Pasteur pipettes to aspirate liquid).
- Remove water until surface is completely dry.
Culturing procedure (done in laminar flow hood under sterile conditions)
- Cut out block of agar and glue it onto the support block of the microtome Vibraslicer (Campden Instruments Ltd.) using super glue.
- When using late embryonic stage (E17-18) mouse fetuses, euthanise dam, remove uterus and free individual fetuses from embryonic sack. Place fetuses into sterile Petri dish and continue as outlined below.
- Decapitate mouse fetus or pup (follow guidelines approved by your Institutional Animal Care and Use Committee).
- Remove skin and skull and place brain onto Whatman filter paper disk in a 60mm petri dish filled with cold DS.
- Cut off the cerebellum using a sterile razor blade.
- Pick up brain using a spatula and drain excess fluid on filter paper, then transfer brain to Vibraslicer support block and glue brain in place (caudal side up and ventral part facing agar).
- Fill chamber with cold DS. Set speed selector to 8-9.
- Cut 200-400 µm sections, beginning from olfactory bulb. Once the blade of the Vibraslicer enters the cortex, begin cutting 600µm coronal sections. An E17-18 mouse brain typically yields 3-4 usable slices, while a P0 mouse brain yields 4-5 usable slices.
- Transfer brain slices to 35 mm petri dish labeled DS 2 using reverse end of un-plugged 10 ml glass Pasteur pipet.
- Dissect out cortex and remove meninges using glass needles pulled from capillary tubes.
- Cut cortical rinds into small pieces, 0.5-1 mm in length.
- Filter ES through 0.2 µm filter attached to a 5 cc syringe into a 35 mm petri dish.
- Transfer the cortical tissue pieces to petri dish containing filtered ES.
- Incubate at 37°C for 30 min.
In the meantime:
- Clean up hood, Vibraslicer and dissecting tools.
- Add 5 ml of warm NBM/B27 into a 60 mm petri dish.
After 30 min. incubation:
- Transfer tissue to 10 ml DS. Let settle for 1 min.
- Transfer tissue to first Hi tube swirls and let settle for 2-3 min.
- Transfer to second Hi. Repeat as above.
- Transfer to first Li. Repeat as above.
- Transfer to second Li. Repeat as above.
- Transfer to third Li. Repeat as above.
- Transfer tissue to the 60 mm dish with media.
Under the dissection microscope:
- Clean debris from tissue and triturate each piece by gently passing through a pulled glass pipet (use decreasing bore sizes) to loosen up the tissue.
- Transfer cell suspension to 15 ml conical tube containing the rest of NBM+B27 (Make a total of 13 ml for a 24 well plate or 11 ml for 5 – 35 mm dishes. Mix gently and wait for large pieces of tissue to settle).
- Transfer cell suspension (0.5 ml/well for a 24 well plate or 2 ml / 35 mm culture dish).
- When using glass coverslips, add 80 µl cell suspension per coverslip and allow 1 hr in tissue culture incubator for cells to adhere before flooding the chamber with more NBM/B27 media.
- Maintain cultures at 37°C and 5% CO2.
Next Day
24 well plate: Feed each well with 0.5 ml non-neuronal cell conditioned NBM/B27 medium (cNBM/B27; Protocol for preparation of cNBM/B27 medium appears below)
35mm dishes: Replace 0.5 ml with cNBM/B27 medium.
Maintain culture by replacing 0.5ml of medium with fresh cNBM/B27 every 2-3 days.
Although the culture media does not promote glia cell proliferation, cultures can be treated with 5µM FDU (5-Fluoro-2’-deoxyuridine, Sigma F0503) at day 3-5 in culture to further reduce the number of glial cells if needed.
SET-UP for Dissection and Culture
Sterilize dissecting tools in 70% EtOH or autoclave them:
- Scissors (med. and small) Spatulas (med. and small)
- Tweezers (med. and small) Razor blade
- Blade for vibraslicer Buffer bath
- Small metal wedge for blade Support for brain
Other materials:
- Petri dishes (60 mm)
- Petri dishes (35 mm)
- Filter paper
- Glass pipet 10 ml
- Sterile disposable pipets, 5,10 and 25 ml
- Syringe filter, 0.2 µm
- Syringe 5 cc
- Centrifuge tubes, 15 ml
- Sterile Pasteur glass pipets
- PDL coated culture dishes or glass coverslips
Label six 15 ml centrifuge tubes and follow preparation:
Tube #1: Enzyme solution:
Add 50 U of papain (Worthington LS 03126) to 5 ml DS containing:
- 100µl of L-cystein (0.8mg)
- 7 µl 0.1N NaOH
- 50 µl APV (5mM)
Leave solution out at room temperature to clear. Note that enzyme solution will appear ‘cloudy’ at first and needs to clear before use.
Tube #2 & #3: Hi Enzyme Inhibitor:
3 ml DS + 300 µl BSA/Ti + 30 µl APV (5mM).
Mix gently to avoid bubbles, then divide into two 1.5ml aliquots.
Tube #4 – #6: Low Enzyme Inhibitor:
8 ml DS + 80 µl BSA/Ti + 80 µl APV (5mM).
Mix gently to avoid bubbles, divide into three 2.6 ml aliquots.
Place tubes numbered #2 through #6 on ice. Leave tube #1 (Enzyme Solution) at room temperature.
SOLUTIONS AND ALIQUOTS
- Solution A (Buffered Saline) Sigma Formula wt 500 ml [conc.]
- Sodium Chloride NaCl S-9625 58.45 80.0g 137mM
- Potassium Chloride KCl P-4504 74.56 4.0g 5.4mM
- Sodium Phosphate Dibasic anhydrous Na2HPO4 S-0876 142.0 0.24g 0.17mM
- Potassium Phosphate Monobasic anhydrous KH2PO4 P-5379 136.09 0.3g 0.22mM
Weigh out all ingredients and mix until dissolve with 400 ml ultra filtered water. Bring final volume to 500 ml. Place in a clean bottle and autoclave. Store at 4°C and label “DS Solution A”.
- Solution B (Hepes) Sigma Formula wt 250 ml [conc.]
- Hepes Hepes H-3375 283.3 20.97g 9.9mM
Add ultra-filtered water up to 200 ml. Mix until dissolved and bring final volume to 250 ml. Place in a clean bottle and autoclave. Store at 4°C and label “DS Solution B”.
- Working Solution 500 ml [conc.]
- Ultra filtered water 400 ml
- Stock solution A 25 ml
- Stock Solution B 14 ml
- D (+)-Glucose Sigma G-8270 3.0 g 33.3mM
- Sucrose Sigma S-0389 7.5 g 43.8 mM
Adjust pH to 7.4 with 1N NaOH. Bring final volume to 500 ml with ultra filtered water. Decant into a clean glass bottle and autoclave. Store at 4°C. Label “Dissecting Solution”.
Poly-D-Lysine Preparation:
- Prepare 10x stock solution of poly-D-lysine (PDL; Sigma P-7280) in sterile H2O at 1mg/ml.
- Make 1.0 ml aliquots. This solution can be stored at -20°C for up to 3 months.
MEDIA
- Add 10ml of B-27 Supplement (Gibco 17504-044) to 500 ml NBM bottle (Neurobasal Medium, Gibco 21103-049).
- Make 40 ml aliquots and store at 4°C.
APV
- Add 10 ml sterile H2O to vial of 10 mg APV (2-Amino-5-phosphonopentanoic acid, Sigma A-5282) and mix thoroughly.
- Prepare 180 µl aliquots and store at -20°C.
BSA/Ti
- Dissolve 1 g BSA (Bovine Albumin. Sigma A-7030) and 1 g Trypsin Inhibitor (Sigma T-9253) in 10 ml DS.
- Adjust pH to 7.4 with 1N NaOH.
- Sterilize by filtering through 0.2 µm syringe filter.
- Divide into 400 µl aliquots and store at -20°C.
L-CYSTEINE
In an Eppendorf tube dissolve 0.6 mg L-Cysteine (Sigma C-7755) in 200 µl DS.
AGAR (4%)
- Dissolve 4 g of Bacto Agar (Difco 0140-01) in 100 ml sterile water. Keep at 4°C until needed for culture.
- Microwave Agar to melt and fill a 35 mm petri dish. Let sit to cool down and polymerize.
PAPAIN (Worthington LS 03126)
NON-NEURONAL CULTURES IN NUNC CULTURE BOTTLES
COATING CULTURE BOTTLE (4 Nunc bottles)
- Add 9 mL sterile water to each of 3 tubes of 10X PDL to make 1X PDL.
- Transfer 30 ml 1X PDL in the first bottle.
- Move slightly until all the growth surfaces are covered. Let sit for 1 min.
- Transfer 1x PDL to second bottle. Let sit for 1 min.
- Repeat with the third and forth bottles.
- Rinse all the four bottles 2X with 150 ml sterile H2O.
PREPARE ENZYME SOLUTION (ES):
- In a 0.6 ml centrifuge tube weight out 0.8 mg L-Cysteine (Sigma C7755), add 150ml DS and vortex until crystals are dissolved.
- Transfer 5 ml DS to a 15 ml conical tube, then add 150ml L-Cysteine.
- Add 50 units Papain. (Worthington LS 03126)
- Add 7ml 0.1N NaOH.
PREPARE MEM (GIBCO # 11090-081)
- Remove 65 ml from the 500 ml MEM bottle. Save 10 ml to prepare Glucose.
- Add 5 ml Pen/Strep (Gibco #15070-063).
- Add 10 ml 1M Glucose (Sigma G-8270) (1.8 g/ 10 ml MEM)
- Add 50 ml Fetal Bovine Serum (Gibco # 16140-071) or Bovine Calf Serum (Omega BC-04).
- Mix well, aliquot and store at 4°C.
NEUROBASAL MEDIUM/B-27 SUPPLEMENT (NBM/B27)
NBM, 500 ml (Gibco #21103-049); B-27 (50X), 10 ml (Gibco #17504-044)
- Add the entire content of B-27 vial to the NBM bottle and mix.
- Aliquot and store at 4°C.
DISSECTION OF BRAIN TISSUE
- Dry dissecting tools from 70% EtOH before dissection.
- Transfer 10 ml DS in each of 3-15 ml tube.
- Select mouse pups (P0 – P3), you will need three brains for 4 bottles.
- Add cold DS to 35 mm petri dish.
- Decapitate mouse pup and dissect out brains.
- Place brains into the petri dish containing cold DS.
- Chop tissue into pieces, small enough to pass through a glass pipette.
ENZYME DISSOCIATION
- Remove as much DS as possible from the dish with a Pasteur pipette.
- Filter ES through a 0.2 µm filter and 5cc syringe.
- Place ES in the dish with tissue.
- Incubate tissue at 37°C incubator (CO2 free) for 30 min.
WASHING TISSUE
- Transfer tissue with glass pipette to the first DS tube, making sure to get as little ES as possible.
- Centrifuge for 30 sec at 1500 rpm.
- Transfer tissue and repeat procedure two more times.
TRITURATION
- Place 38 ml of MEM/FBS into a 50ml centrifuge tube
- Add 3 ml of MEM/FBS to a 35 mm dish and transfer tissue to this petri dish.
- Dissociate the tissue by gently triturating it through a 1000µl eppendorf pipet tip.
- Transfer cell suspension to the tube containing 38ml of MEM/FBS and mix well.
PLATING
- Stand the culture bottles upright to get equal amounts of the cell suspension and media in each bottle.
- Put 90 ml of MEM/FBS in each culture bottle.
- Add 10 ml of cell suspension in each bottle.
- Label: Non-Neuronal, initials and date. Incubate at 37°C and 5% CO2.
FEEDING
- On Day 3 or 4, feed cells with warm MEM/FBS (decant all the media and replace it with 100 ml of MEM/FBS). Cultures may take 7-10 days to become confluent.
- On Day 7-10 change the media for NBM/B27.
- Next day, collect the media. Filter with 0.22µm
- Make aliquots of 40 ml and feed culture with MEM/FBS.
- Next 2 or 3 days, change media for NBM/B27
- Next day, collect and repeat the procedure.
- Keep collecting media for approx. 2 weeks.