Cultures

Hippocampal Culturing Protocol from Nunez

NOTE: Glial-treated medium and coverslips must be prepared prior to culture day.

1. Sacrifice DAM.

2. Spray down abdomen with 70% EtOH.

3. Cut back skin and fur and spray down muscle and tools with EtOH.

4. Cut distal to the ovaries.

5. Remove the uterus by lifting at both ends while cutting it free (spray it down with EtOH).

6. Transfer fetuses to a sterile Petri dish (on ice).

7. Remove fetuses from uterus and place them in small Petri dish (a few per dish) with HBSS+.

8. Dissect out hippocampi and place in 2 or 3ml HBSS+ in a 15 ml tube.

9. Fill HBSS to 4.5 ml.

10. Add 0.5 ml trypsin.

11. Incubate 15 min at 37 degrees.

12. Remove HBSS+/trypsin by pipette, being careful not to disturb the hippocampi settled at the bottom.

13. Add 5 ml of HBSS+ and gently shake.

14. Let sit at 37 degrees for 5 min.

15. Repeat steps 11 and 12.

16. Add 4.5 ml of HBSS+ and 0.5 ml of DNAse (Sigma# DN25).

17. Pipette up and down vigorously until homogeneous. DO NOT FROTH.

18. Combine the following and allow to sit for 4 min:

a. 10 ul Trypan Blue

b. 180 ul HBSS+

c. 10 ul cell suspension

19. Determine cell count and viability using a hemacytometer (clear is alive, blue is dead). (Average # of living cells counted on a 4X4 grid)(10000)(20) = # of cells/ml

20. Determine the volume of cell mixture needed to plate at the desired density.

21. Pipette the determined amount of cell suspension to dishes containing Plating Media. Swirl the dishes to disperse cells. Make sure coverslips do not overlap.

22. Allow the cells 2-4 hours to plate. Check to see that cells are plated.

23. Transfer the coverslips neuron-side-up to dishes containing Glia-treated Neuronoal Media.

Replace 1/3 of the medium at least weekly and supplement experimental treatments as needed.

3 ml neurobasal to small dishes (35 mm) at 37 degrees.

10 ml Plating to coverslips (60 mm) 37 degrees.

Cortical and Hippocampal Cultures

Coating the plates:

1. Prepare stock solution: 5 mg/ml PDL from Sigma(Cat # p-1024). Sterilize by filtering through 0.2um membrane (it is difficult to filter because the solution is very sticky).

2. Store stock solution at -20.

3. Coat Falcon or Costa (Costa is better) plate with 150 ug/ml PDL at 37 degrees for at least 3 hours (overnight is better). Use about 0.4-0.5 ml per well for a 12-well plate.

4. Wash with sterilized water for at least 2 times, shortly before culturing.

Make dissociation media (DM)

Prepare 100 ml for one litter of rat.

For 100 ml DM:

84 ml water

8.18 ml 1M Na2SO4

6.0 ml 0.5M K2SO4

0.58 ml 1M MgCl2

0.252 ml 0.1M CaCl2

0.15 ml 1M HEPES, pH: 7.6

0.8 ml 2.5M Glucose, or 360 mg powder

0.2 ml 0.5% phenol red

Adjust pH to 7.6 (use pH meter). Store on ice.

Make digestion buffer:

For each litter of rat, make 10 ml. Add 100 units (or 10 units/ml) papain (Worthington Pap.3126) and 0.45 mg/ml cysteine in DM, adjust pH to 7.6. Store on ice. Warm up in 37 degree water for 20 min (before digesting brain bits), add 10,000 units DNase I (1 vial) (Roche, Cat #: 04536282001. Obtainable from Biochem store. There are 2 vials in the package, each containing 10,000 units. Put 1 ml of digestion buffer in the vial, dissolve the powder, and transfer the solution back), filter sterilize, and use for digestion.

Dissection:

Dissect hippocampi and the dorsal part of the cortex, and put them in ice-cold DM. Remove meninges, and chop into small bits (little bits smaller than 1 cubic mm) in digestion buffer. Use 3 ml for hippocampi (collected from one litter of rat), and 7 ml for cortex (collected from 4 to 5 rats).

Digestion:

Digest brain bits in digestion buffer (use 50 ml cornical tube) at 37 degrees for 30 to 40 min. Shake gently ever 5 to 10 min.

Washing and plating:

After digestion, wash tissue with DM (room temperature) for 2 to 3 times, triturate in Neurobasal A (room temperature) using 10 ml pippet for 20 to 30 times (up and down counts for one). When triturating, avoid bubbles and avoid high speed pipetting. Let the tissue settle for 1 to 2 min, transfer the supernatants (containing neurons) to a new 50 ml tube. Note: there will still be significant tissue chunks left after trituration, and it is okay. Mix 20ul cell with 20 ul 0.4% trypan blue. Count the cells. Cell density = 20,000 X the number in 16 small squares.

Plate 0.8 to 1 million cells in one well of the 12-well plate. I normally adjust cell density to 0.8 to 1 million/ml and dispense 1 ml to each well. If yield is low, 0.5 million/well will also give dissent density.

One to 2 hours after plating, change Neurobasal A to culture media (Neurobasal A, 1X B27, 1X pls, 0.5 mM Glutamine).

Normally, 1 litter of 13 rats will yield 20 million hippocampal neurons. And cortex from 5 rats will yield 6 million neurons total.

Cortical Culture (Adopted from Isabella Graf)

Solutions:

Poly-Lornithine (Sigma 0.01% solution: P4957), dilute to 15 ug/ml in ddH2O (1:6.67 or 39.7 ml H2) + 7 ml Poly-ornithine stock

1 ug/ul fibronectin in PBS (bovine, Sigma F4759

1 mM Progesterone: (Store at 4C)

1 g Progesterone

3180 ml ethanol

Transferrin: (Store at 4C)

100 mg Transferrin

25 ml H2O

Medium:

Neuralbasal medium

HBSS + 30 mg/l NaHCO3

HBSS + 20% FCS + 350 mg/l NaHCO3

Trypsin/DNAse Solution:

Add 20 mg trypsin to 10 ml of HBSS solution

Add 20 ul of DNAse (200u/ul H2O) to the above solution

Trypsin: Aldrich-Sigma T1005, $156.40/1g, $643.30/5g

DNAse Type IV: Sigma D5025, $278/375,000 units

Coverslip Preparation:

Use 12 mm round glass coverslips, thickness 0. Assistant is a good brand.

Place coverslips in a container with a lid. Rinse twice with acetone, and once with ethanol and once with 70% ethanol. Rinses should be at least 1 hour each, but can go overnight.

Dry coverslips by separating them and placing them on clean paper towels in a laminar flow hood or fume hood. Collect clean, dry coverslips in a glass petri dish and autoclave to sterilize. Undried coverslips can be stored in the rinsing container in 70% ethanol.

Siliconized Pipets Preparation:

Submerge pasteur pipets point down in siliconized petroleum ether (0.2% Silicon Oil, Adrich Chemical Co. cat no 14615-3 (500 g $32.60) added to Petroleum ether (Aldrich Chemical Co. cat no. 30031-4 (1 L $30.15)). After pipets are coated (about 1 hour), pour the solution back into the bottle for re-use. Turn the pipets point upwards and rinse them in running water until the smell subsides (about 4 hours). Dry the pipets, plug the ends with cotton, and autoclave to sterilize.

Coat Dishes or Coverslips: Poly-Lornithine (Sigma 0.01% solution: P4957) dilute to 15 ug/ml in ddH2O (1:6.67 or 39.7 ml H2) + 7 ml Poly-ornithine stock. For 1 hour.

Wash 3x with ddH2O (or PBS).

Coat with 1 ug/ul fibronectin in pBS (bovine, Sigma F4759). One can leave it ON at 4C after coating, 1 hour, wash 3X with PBS. Need to be pretty dry at the end so that cells stay on the coverslip.

Dissection: dissect in DMEM/F12 Medium

1. Cut the hemispheres away from the rest of the brain. Turn them upside down and start to peel the meninges away carefully without tearing at the cortex. Then unroll the cortex and cut it away from the underlying brain areas. At this point, you should have a thin flap of tissue that does not show any blood vessels. Transfer with a transfer pipet to a fresh dish with medium on ice.

2. After you are done with all embryos, transfer the cornices into a small dish with HBSS (CMF) + 20% FCS + 350 mg/l NaHCO3 and cut them into small cubes.

3. Transfer cubes into a 15 ml corticle and wash 3x with HBSS + NaHCO3 (2-10 ml/wash). For the washes, add solution, let pieces settle, and aspirate supernatant.

4. Add 10 ml Trypsin/DNAse solution, sterilize using a syringe filter.

5. Incubate 4-5 min. at room temperature and occasionally shake gently. Aspirate supernatant and add 10 ml (2 ml X 2 if using knockout mice) of HBSS 20% FCS and repeat the washes as before in 3. (3X HBSS + NaHCO3)

6. Add 20 ul of DNAse to 2 ml of HBSS and filter on to cells (adjust depending number of cells, e.g. 10 ml HBSS + 100 ul DNAse). Mechanically dissociate by pounding.

7. Add 3 ml of HBSS/20% FCS. Centrifuge 10 min. at 1000 rpm and 4 degrees C.

8. Resuspend the pellet in 0.5 ml culture medium.

9. Count the cells: Take 5 ul + 45 ul HBSS + 50 ul trypan blue. Count the cells in the square (n). n x (20 x 10^4). Dilute the cells to 2x10^6/ml.

10. Add 25 ul (5x10^4) cells to the middle of the coverslips. Let the cells adhere for 1 hr in incubator and add medium (0.75-1ml/24 well). (For 96 well plates, 5x10^4 cells/well).

Dissociated Cultures of Cerebellar Neurons

Notes:

- Can do on counter (i.e. in non-sterile conditions).

- Use P6-P8 rat pups, or P5 mouse pups

- Keep on ice whenever possible.

- For each step, do all pups, before moving to next step.

1. Preparation

- Prepare plates (coat and wash, leave in incubator until needed)

- Get two ice-bucket with ice

- Make CbC media; place on ice, warm up at 37 degrees C water bath 20 min before needed.

- Add cold HHGN to 1 of 35 mm petri-dish, 2 of 60 mm petri dishes for dissection, keep dishes on ice. Keep the HHGN bottle on ice for later use.

Note: no need to use TC dishes for dissection. Use petri dishes instead for cost reasons.

- Sterilize tools by soaking several minutes in 75% EtOH (in tray): large forceps, 2 fine forceps, curve forceps, 1 small and 1 large scissors; after soaking, let air dry.

- Remove one tube each of TDn and DnB from -80 degrees C; place at room temperature to thaw.

2. Isolation of Cerebella ("Cb")

- Ethanol spray animal head.

- Cut off heads into plate on ice.

- Transfer one head at a time to another 10 cm petri dish on bench.

- Hold nose with large forceps, insert small scissors into hole at base of skull; cut with scissors through skin and skull, from side of neck to top of head, across, and back to neck. (Don't cut too deeply, so as to not cut brain itself.)

- Remove flap of skin, skull, from front to back, being careful not to let brain fall out.

- Pinch off cerebellum with fine forceps, into 60 mm plate containing HHGN (on ice).

3. Removal of Meninges

- Do one Cb at a time under dissecting scope, not on ice.

- Pull away meninges using two fine forceps; not necessary to remove entirely.

- Remove as much meninges as possible, but do not take too much time. The faster the dissection, the healthier the cultures will be.

- Transfer the "clean" Cb to the 35 mm petri-dish with HHGN on ice.

Note: 5 min before finishing dissection, place TDn at 37 degrees C H2O bath!!!

4. Processing, Plating, and Feeding of Cerebella

Note: In tissue culture hood; keep sterile. From this point on, leave at room temperature.

- Pour Cb's into 50 ml tube (do not pipet, so as to not break up).

- Rinse 3 times with 3 ml HHGN.

- Digest for 15 min at room temperature, in 5 ml TDn for 5-7 pups, or 10 ml for more pups (lower volume OK if doing individual Cb).

Note: at this time, place DnB, OptiMEM, and Cb growth medium at 37 degrees C H2O bath to warm up!!!

- Remove soup, wash 3 times with 5-10 ml HHGN.

- Add 5 ml DnB

- Triturate with 5 ml plastic pipet until homogeneous (25 times)

- Let settle 5 minutes to remove clumps, transfer supernatant to second tube.

- Re-triturate the settled clumps with 5 ml DnB (more vigorously; seal pipet tip, triturate 15 times).

- Let settle 5 minutes; transfer/pool supernatant with first supernatant.

- Centrifuge 500-800 PRM, 3 minutes in room temperature

- Resuspend pellet in 10 ml culture media (should resuspend easily) (It is normal for supernatant to be cloudy at this point. If single cerebellum, resuspend in 3 ml.)

- Dilute cell suspension into a sterile TC bottle with appropriate volume of OptiMEM/glucose to estimated 1 x 10^6/ml: one Cb gives about 10-15 x 10^6/ml cells, so if you have 10 Cb, dilute to about 100 ml total.

- Count alive cells - cells with shiny cell bodies. (Mix cells well. Add 10-15 ul cell suspension to each side of the hemacytometer. The cells should be evenly distributed in all grids. If you have big discrepancy, mix cells and count again. count 2-4 corners of grid from each side, then average them. Calculate cell concentration: average cell number in each grid x 10^4 = cell concentration. Expected results: 50% live cells, 10-15 x 10^6 cells per Cb.)

- Seed to plates/wells: Mix diluted cell suspension in the bottle frequently during seeding, so cells do not settle. (Mix diluted cell suspension in the bottle frequently during seeding, so cells do not settle. (24 well: 5 x 10^5, 0.5 ml; 35 mm: 2.5 x 10^6, 2-3 ml; 60 mm: 5 x 10^6 3-5 ml; 100 mm: 1.5 x 10^7, 10-15 ml). After plating, shake plates left-right, top-bottom, before and after placing into incubator, to evenly distribute cells.

Note: Tips to avoid stagging converslips: Add cell suspension to 5 plates at a time, push down coverslips to plates with a pasteur pipette or pipet tip, shake plates left-right, top-bottom for even seeding, and repeat the process until all the cells are seeded.

- Change media 1-2 hours after seed (3 ml culture media).

- Grow in 5% CO2 at 37 degrees C.

- On 1 DIV (approx 24 hr), add araC to 10 uM (araC: -80 degrees C, 10 mM stock).

- Feed cells: with glucose on 3-4 DIV (add glucose to final conc. of 0.8 mg/ml), feed on approximately 8 DIV, and weekly thereafter. Alternatively, can change 1/2 medium with fresh medium containing AraC every 2-3 days.

5. Transfection: Can be performed on DIV 3-4

6. Death Induction

A. Serum-Withdrawal: On 6-7 DIV

Wash 2 times with KI (24 well: 0.5 ml, 35 mm: 2 ml, 60 mm:3 ml)

Add KI to induce death, conditioned media or +KI to promote survival.

B. Glutamine treatment: on DIV 8-9

Primary Neuron Culture

1. Coat plates with 50-100 ug/ml poly-D-lysine (or poly-L-lysine) in water. Incubate plates in 37C incubator or at room temperature for at least 2 hours. 3-4 hours of coating is typical. Overnight is okay too.

2. Prepare dissociation medium/Kvn.Mg (DMKM) solution (For 50 ml DM: 41.94 ml H2O, 4.09 ml 1 M Na2SO4, 3.0 ml 0.5 M K2SO4, 0.29 ml 1 M MgCl2, 0.126 ml 100 mM CaCl2, 0.075 ml 1 M HEPES, pH 7.6, 0.40 ml 2.5 M glucose, and 0.10 ml 0.5% phenol red. For 100 ml 10X Kyn/Mg: 189.25 mg kynurenic acid, 0.2 ml phenol red, 0.5 ml 1M HEPES, 10 ml 1M MgCl2, and water).

3. Prepare papain solutions (10 units/ml Papain suspension and 0.45 mg/ml cysteine. HCl in DM. Adjust pH to 7.4 with NaOH. Filter sterilize. Incubate at 37C for 20-30 minutes before use. Make 5-10 ml per prep), adjust pH to 7.4 with NaOH, sterile filter and keep the tubes on ice.

4. Dissect pups by decapitation. Sterilise the scalp with ethanol. Slit open the scalp and skull with a razor blade. Scoop out the brain with a spatula. Place it on a piece of filter paper saturated with DMKM solution and cut out the appropriate brain structure. Keep the tissue in DMKM until the dissection is done.

5. Warm up papain solution 30 minutes before finishing dissection.

6. Transfer tissues to a sterile 15 ml tube. Allow tissues to settle to bottom of tube. Draw off the supernatant.

7. Digest tissues with 2-10 ml papain solution, depending on the size of the tissue, at 37C, 30-45 minutes with intermittent gentle shaking and swirling.

8. While enzyme treatment is going on, aspirate lysine solution and rinse plates with water.

9. Let tissues settle. Draw off the supernatant and wash the tissues 5 times with 1-2 vol. Neurobasal each.

10. Transfer tissues in 5 ml Neurobasal to a 50 ml tube. Pipette up and down 10-15 times using a 5 ml disposable pipette. Add 5 ml more of neurobasal to the tube, swirl, and let the tissues settle. Transfer the supernatant to a fresh tube. Repeat the trituration steps and pool the supernatant.

11. Do a third round of trituration if necessary. Let the big clumps settle and pool the supernatant with cells collected in step 10. Do cell counting.

12. Dilute cells and plate out. Shake plates in the North-South-East-West fashion to evenly disperse cells (optional). Incubate plates at 37C with 5% CO2.

13. Let cells adhere 1-2 hours. Replace with fresh GM. In general, cultures are ready for assay in 7-14 days.

14. The next two steps are optional depending on application. The next day, add araC (final concentration 5 uM) to the plates to inhibit proliferating cell growth.

16. Change 1/3 volume of GM and replace with GM (KCl-containing for cerebellar) once every 3-4 days if intending to maintain culture for weeks.

Keywords

Adenylyl cyclase (AC), amyloid precursor protein (APP), Alzheimer’s disease (AD), brain derived neurotrophic factor (BDNF), cAMP, cAMP-responsive element (CRE), CREB, extra-cellular regulated protein kinase (ERK), FE65, long-term potentiation (LTP), long-term depression (LTD), memory formation, neuroplasticity, local protein synthesis.