TITLE: Postnatal ventral midbrain dopamine neuronal culture protocols
AUTHORS: Dave Sulzer, Ellen  Kanter

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol details the specific steps and media solutions required to dissect and triturate the postnatal netral midbrain dopamine neuronal cultures on mice.</div></div>

[STEPS]
?. [Aclar Dishes for Electron Microscope Preparations]
Cut Aclar sheets into squares ~ 1.5 cm and drop into Nanopure dd H2O for .
?. [Aclar Dishes for Electron Microscope Preparations]
Swirl the squares in a beaker (if they don’t all immediately float to the top).
?. [Aclar Dishes for Electron Microscope Preparations]
Rinse twice in Nanopure dd H2O for 5 min:
?. [Aclar Dishes for Electron Microscope Preparations]
Expose to 70% ethanol in Nanopure dd H2O for .
?. [Aclar Dishes for Electron Microscope Preparations]
Rinse twice in Nanopure dd H2O for 5 min:
?. [Aclar Dishes for Electron Microscope Preparations]
Expose to  in Nanopure dd H2O for .
[HCl]
?. [Aclar Dishes for Electron Microscope Preparations]
Rinse again several times in water.
?. [Aclar Dishes for Electron Microscope Preparations]
Maintain in 70% ethanol, covered.
?. [Aclar Dishes for Electron Microscope Preparations]
Sylgard the squares to the dishes after the holes are prepared:
?. [Aclar Dishes for Electron Microscope Preparations]
Wearing gloves, prepare about  in a large weighing boat by combining 10 parts resin with 1 part catalyst (by weight) and stirring extensively with flame-sealed Pasteur pipette. Pour contents into new weighing boat to avoid use of resin without catalyst.
[Sylgard]
?. [Aclar Dishes for Electron Microscope Preparations]
Dip an empty 15 ml Falcon tube or glass scintillation vial upside-down into the Sylgard, using it as a rubber stamp to make a circle of Sylgard around the hole, on the outside of the dish.
?. [Aclar Dishes for Electron Microscope Preparations]
Place the Aclar sheets on the circle of Sylgard, covering the hole.
?. [Aclar Dishes for Electron Microscope Preparations]
Allow Sylgard to spread (about ).
?. [Aclar Dishes for Electron Microscope Preparations]
To cure the Sylgard, place dishes, upside-down, at , or at room temperature for 2 to 3 days (cured Sylgard is no longer sticky to the touch).
37 °C
?. [Aclar Dishes for Electron Microscope Preparations]
Store dishes in zip lock bags: initial and date.
?. [Aclar Dishes for Electron Microscope Preparations]
Coat with PDL:
?. [Aclar Dishes for Electron Microscope Preparations]
Add  of the  to each dish. Expose for - at  in a culture hood without UV light.
[40 µg/ml PDL solution]
0 Room temperature
?. [Aclar Dishes for Electron Microscope Preparations]
Rinse 3 times for 5 min each in tissue culture water: Rinse 1/3: rinse in tissue culture water for .
?. [Aclar Dishes for Electron Microscope Preparations]
Remove water and expose to UV for  to sterilize.
Note that these dishes must be coated with laminin before use, just as we do with glass coverslips (see glia dissection protocol)
Left over sylgard from dish preparation can be used to make sylgard circles.
?. [Glass Coverslip Preparation]
Drop 100 coverslips (15 x 15 mm) one-at-a-time into a 200 ml beaker containing . (Leave approx.  at least)
[95% EtOH]
?. [Glass Coverslip Preparation]
Prepare a solution of poly-d-lysine in “nanopure” or tissue culture grade water. The final concentration should be  and 250mL are adequate for 100 coverslips. Place the poly-Dlysine solution in a glass dish of enough area to give 3/4”/2 cm height to the poly-D lysine solution. (the walls of the dish should be at least 1.5”/4 cm, the area no less than 40”/100 cm square)
?. [Glass Coverslip Preparation]
Throw (pour) away excess EtOH. Place ethanol soaked cover slips on to a paper or glass container.
?. [Glass Coverslip Preparation]
Individually flame dry the coverslips (one pass through flame); use forceps.
?. [Glass Coverslip Preparation]
After putting the slip through the flame, allow it to cool in the air in forceps for approximately  while moving back and forth (cracking or sizzling sounds mean it did not cool enough).
?. [Glass Coverslip Preparation]
Put the coverslip into the poly-D-lysine solution. Repeat for all slips.
?. [Glass Coverslip Preparation]
Let sit for , then pour off the poly-D-lysine, then let slips dry ( in the hood with the blower on).
?. [Glass Coverslip Preparation]
Prepare dishes with 10 mm holes punched with a bench punch for wells where neurons and glia will grow. Sylgard the coverslips to the dishes after the holes are prepared.
?. [Glass Coverslip Preparation]
Wearing gloves, prepare about  in a large weighing boat by combining 10 parts resin with 1 part catalyst (by weight) and stirring extensively with flame-sealed Pasteur pipette. Pour contents into new weighing boat to avoid use of resin without catalyst.
[Sylgard]
?. [Glass Coverslip Preparation]
Dip an empty 15 ml Falcon tube or glass scintillation vial upside-down into the Sylgard, using it as a rubber stamp to make a circle of Sylgard around the hole. Use caution when applying the Sylgard as too much will result in excess Sylgard covering the culturing surface of the coverslip whereas inadequate Sylgard will result in a leaky well.
?. [Glass Coverslip Preparation]
Place the coverslips on the circle of Sylgard, covering the hole.
?. [Glass Coverslip Preparation]
Press down on coverslip gently using forceps to eliminate bubbles.
?. [Glass Coverslip Preparation]
Allow Sylgard to spread (about ).
?. [Glass Coverslip Preparation]
To cure the Sylgard, place dishes, upside-down, at , or at room temperature for 2 to 3 days (cured Sylgard is no longer sticky to the touch). Once the dishes are cured they can be stored in zip-lock bags at  until needed.
37 °C
4 °C
Note that these glass coverslips must be coated with laminin before use (see glia dissection protocol
Left over sylgard from dish preparation can be used to make sylgard circles.
If these coverslips ever become unavailable, the Bellco #2 photoetched coverslips have been recommended
?. [The TechTip Preparation]
Obtain a box of 1000 ul pipette tips (unfiltered). Wear gloves.
These are used for triturating cells
?. [The TechTip Preparation]
Flame all tips in order to seal off hole (we recently started using tips without sealing the hole, making additional holes with needles as described below).
?. [The TechTip Preparation]
Using a needle, poke a hole as close to the tip as possible through one side and out the other. This will result in two holes. The purpose of piercing right through both sides is to prevent cells getting caught in the pipette. Rather, they will have an outlet hole. Make 3 types of tips, with Large (21G 1½), Medium (22G 1½) and Small (26G 1½ ) size holes.
?. [The TechTip Preparation]
Autoclave as usual.
?. [Sylgard Circles]
Weigh out the Elastomer Base and the Elastomer Curing Agent. Mix in the ratio 10:1
For use during dissection as a platform to do the cutting:
?. [Sylgard Circles]
Mix well and pour into 12 micro well plate, about 3-4 mm in height.
?. [Sylgard Circles]
Cure in oven at  for  or at  for 5-7 days.
60 °C
60 Room temperature
?. [Sylgard Circles]
Cut circles out of wells using razor and individually wrap each circle in aluminum foil, autoclave to sterilize.
Left over sylgard from dish preparation can be used to make sylgard circles.
?. [Glia Preparation on Coverslip Petri Dishes]
LAMININ COATINGBefore the dissection/digestion time, dilute 1 aliquot of laminin (140ul of 0.5mg/ml laminin = 70 µg) in . Final concentration .
[sterile MEM]
?. [Glia Preparation on Coverslip Petri Dishes]
Prepare the papain dissociation solution. Each vial should hold tissue from a maximum of 2 animals though it’s better to digest 1 rat cortex per vial.
?. [Glia Preparation on Coverslip Petri Dishes]
Sterilize the top of each Nunc specimen vial (25 ml) with ethanol. Punch two holes in the top with an 18 gauge needle, using a separate needle to bore holes. In one hole place a new needle with a 0.22 µm filter attached. Leave the other hole empty for ventilation.
?. [Glia Preparation on Coverslip Petri Dishes]
Add an HCl-cleaned, autoclaved micro-stir bar to the vial. Fill each tube with - (sterile filtered with 0.22 µm filter). Place in temperature controlled   water bath with magnetic stirrers.
5 mL
[freshly made papain solution]
37 °C
?. [Glia Preparation on Coverslip Petri Dishes]
Arrange dissection tools, microscope, dissection light, a bunch of transfer pipettes, sterile Petri dish with Sylgard circle, beaker, and 70% ethanol in a 50 ml tube in the sterile hood.
?. [Glia Preparation on Coverslip Petri Dishes]
Prepare two buckets of ice to chill the PBS and for the pups.
?. [Glia Preparation on Coverslip Petri Dishes]
Cut aluminum foil squares (about 15 x 15 cm).
?. [Glia Preparation on Coverslip Petri Dishes]
Perfuse dissociation tube (papain tube) with a steady flow of humidified carbogen (95% oxygen, 5% CO2) delivered through the filter/needle (flow rate adjusted to about 1 bubble per second).
?. [Glia Preparation on Coverslip Petri Dishes]
When the papain has reached a pH of -  in about  -  (indicated by a red color; purple indicates that the papain solution is too alkaline and orange that it is too acidic), anesthetize pups and begin dissection.
?. [Glia Preparation on Coverslip Petri Dishes]
Anesthetize pups with an intraperitoneal injection of ketamine (0.01 and 0.05 ml of 100 mg/ml for mouse and rat pups, respectively).
?. [Glia Preparation on Coverslip Petri Dishes]
When pups begin to show sedation, put them on ice till hypothermic.
?. [Glia Preparation on Coverslip Petri Dishes]
Rinse aluminum foil square, scissors and head with 70% EtOH.
?. [Glia Preparation on Coverslip Petri Dishes]
Decapitate pup, allowing head to fall onto aluminum foil square and move to hood.
?. [Glia Preparation on Coverslip Petri Dishes]
Remove the olfactory lobes and brain stem (with V-cut). Hemi-sect brain and then peel off meninges. Shell out cortex, taking care to remove the hippocampus. Remove any torn areas to avoid DNA release. Cut into 1 mm3 chunks.
?. [Glia Preparation on Coverslip Petri Dishes]
DIGESTIONPlace chunks in papain as with neuronal prep, without kynurenate (1 cortex/vial). Chunks will be digested enough to triturate during the initial  - . We judge by the appearance of the chunks (like cotton candy) when they are ready.
?. [Glia Preparation on Coverslip Petri Dishes]
DISSECTIONObtain one or two P1-P3 rats.
?. [Glia Preparation on Coverslip Petri Dishes]
Add at least per round well (enough to cover the surface) for minimum  at  (can be done O/N at +4°C) before plating cells and leave in flow bench. Aspirate off laminin and wash each well with MEM. Place into  incubator to warm dishes before plating the cells. 1 aliquot is enough for about 50-60 dishes.
[10 µg/mL laminin ]
60 Room temperature
37 °C
?. [Glia Preparation on Coverslip Petri Dishes]
TRITURATION AND PLATINGWarm up  for rinsing and trituration.
[ sterile M10C-G]
?. [Glia Preparation on Coverslip Petri Dishes]
From this point forward, everything should be done in a sterile laminar hood with sterile technique.
?. [Glia Preparation on Coverslip Petri Dishes]
Remove chunks from enzyme vial using transfer pipette and place in 15 ml conical test tube.
?. [Glia Preparation on Coverslip Petri Dishes]
Rinse chunks 3 times using , allowing them to settle each time and discard supernatant:
[M10C-G]
?. [Glia Preparation on Coverslip Petri Dishes]
Using  triturate up to a maximum of 10 times (3-5 times should be sufficient) using a regular 1 ml pipette tip.
[warm M10CG]
?. [Glia Preparation on Coverslip Petri Dishes]
Remove supernatant to a separate tube, and replace the volume with .
[fresh M10C-G]
?. [Glia Preparation on Coverslip Petri Dishes]
Repeat trituration using the tech tips with smaller holes each time until the chunks have all fallen apart.
?. [Glia Preparation on Coverslip Petri Dishes]
Pellet the glial cells at .
Centrifuge: 1000 33, 5 min
?. [Glia Preparation on Coverslip Petri Dishes]
. Resuspend pellet in , and count the cells using hemocytometer
[M10C-G]
?. [Glia Preparation on Coverslip Petri Dishes]
Dilute to plating density of 1,000,000-1,500,000 cells/ml.
?. [Glia Preparation on Coverslip Petri Dishes]
Remove laminin from wells and plate about 80,000 cells per well. Plate by dripping the cell suspension from about 10 cm above the dish to help minimize number of surviving neurons in glial prep.
?. [Glia Preparation on Coverslip Petri Dishes]
Tap the sides of the trays (not for island cultures) in order to evenly distribute cell suspension.
?. [Glia Preparation on Coverslip Petri Dishes]
Allow cells to settle for -  in incubator.
For island preps allow 2-5 hours.
?. [Glia Preparation on Coverslip Petri Dishes]
Chill M10C-G in meantime.
?. [Glia Preparation on Coverslip Petri Dishes]
RINSING AND FEEDINGFor monolayers: squirt  into each well, moving the pipette around in a circular motion to wash each well evenly.
[cold MEM]
?. [Glia Preparation on Coverslip Petri Dishes]
Aspirate off MEM and repeat wash:
?. [Glia Preparation on Coverslip Petri Dishes]
For island cultures: One dish at a time, lift the cover and tilt the dish at a 45° angle with the hood countertop. Using a transfer pipette very gently, drip onto a spot on the dish just above the well so that it slides down the incline and over the well.
[cold MEM]
?. [Glia Preparation on Coverslip Petri Dishes]
Aspirate off the MEM.
?. [Glia Preparation on Coverslip Petri Dishes]
Feed with .
[cold M10C-G]
?. [Glia Preparation on Coverslip Petri Dishes]
Add FDU to the dishes when cells are ~70% confluent (about 3-5 days). If neurons are still present at this point, remove the medium and cold-feed with  before adding FDU.
[M10C-G]
?. [Glia Preparation on Coverslip Petri Dishes]
Astrocytes dishes should be at least one week old and at most 4 weeks old at the time the neurons are plated. Feed with cold M10C-G at one month if not already used.
?. [Neuronal Preparation on Coverslips with Glia Monolayer]
Please see "Before Starting" in Guidelines & Warnings section on Things to Prepare during the Week before Plating Glia and Neurons.
?. [Neuronal Preparation on Coverslips with Glia Monolayer]
First, turn on the dissociation water bath and let warm to .
37 °C
?. [Neuronal Preparation on Coverslips with Glia Monolayer]
Make the papain dissociation solution (see Media section). Each vial can hold tissue from 1 to maximum 10 mice preps.
?. [Neuronal Preparation on Coverslips with Glia Monolayer]
Sterilize the top of each Nunc specimen vial with EtOH. Make two holes in the top with an 18 gauge needle, using a separate needle to bore holes. In one hole place a new needle with a 0.22 µm filter attached. Leave the other hole empty for ventilation.
?. [Neuronal Preparation on Coverslips with Glia Monolayer]
Arrange dissection tools, microscope, dissection light, a bunch of transfer pipettes, sterile Petri dish with sylgard circle, beaker, and 70% ethanol in a 50 ml tube in the sterile hood.
?. [Neuronal Preparation on Coverslips with Glia Monolayer]
Get 2 buckets of ice to chill the PBS and to anesthetize the pups.
?. [Neuronal Preparation on Coverslips with Glia Monolayer]
Rip aluminum foil squares (about 15 cm2 ).
?. [Neuronal Preparation on Coverslips with Glia Monolayer]
Fill “insulin” syringe with 100 mg/ml ketamine solution.
?. [Neuronal Preparation on Coverslips with Glia Monolayer]
Obtain as many P1-P3 rat or mouse pups as needed (animals which are one to three days old)
?. [Neuronal Preparation on Coverslips with Glia Monolayer]
Superfuse dissociation tube (papain tube) with a steady flow of humidified carbogen delivered through the filter/needle (Flow rate adjusted to about 1 bubble per second).
?. [Neuronal Preparation on Coverslips with Glia Monolayer]
When the papain has reached a pH of  - in about - (indicated by the red color, not too purple, not too orange), anesthetization and dissection of pups can begin.
?. [Neuronal Preparation on Coverslips with Glia Monolayer]
DISSECTIONAnesthetize pups with an intraperitoneal injection of ketamine (0.01 ml and 0.05 ml of 100 mg/ml ketamine for mouse and rat pups, respectively).
?. [Neuronal Preparation on Coverslips with Glia Monolayer]
When pups begin to show sedation, put them into ice till hypothermic.
?. [Neuronal Preparation on Coverslips with Glia Monolayer]
Rinse aluminum foil square, scissors and head with 70% EtOH.
?. [Neuronal Preparation on Coverslips with Glia Monolayer]
Decapitate pup, allowing head to fall onto aluminum foil square and move to hood.
Some types of mouse tails (for ex, VMATs) need to be saved for genotyping. Prepare a numbered 1.5ml tube for each mouse. After the mouse has been decapitated, remove the tail using a fresh, sterile razor blade and place tail in numbered tube. Put all tubes on ice during the dissection. After dissection, store tubes in -80°C freezer. Alternatively, number the pups with the marker on their back, cut the tails and genotype the pups before the cultures, and no later than 1- day old. This way the pups with the same genotype can be cultured together rather than individually.
?. [Neuronal Preparation on Coverslips with Glia Monolayer]
Gently remove brain into ice cold PBS-filled Petri dish as described for glia. With anterior of brain facing left on the SYLGARD® circle (dissection will be described for right handed people) use a fine forceps in the left hand to pin the two hemispheres of the brain to the SYLGARD®. Remove any remaining meninges.
?. [Neuronal Preparation on Coverslips with Glia Monolayer]
Place the brain, dorsal side down, anterior facing left onto the SYLGARD®. Using forceps, pin the brain to the SYLGARD® to immobilize.
?. [Neuronal Preparation on Coverslips with Glia Monolayer]
Set the magnetic stirrer on low. Chunks should swirl around in the vial gently.
?. [Neuronal Preparation on Coverslips with Glia Monolayer]
Digest for approx. - or until chunks have fallen apart and look something like cotton candy. Periodically examine the tubes to see if segments are sticking to the stir bars; if so, tap vials to loosen tissue.
?. [Neuronal Preparation on Coverslips with Glia Monolayer]
DISH AND MEDIA PREPARATIONOn day before dissection, take glia dishes and wash them twice with :
[cold MEM]
?. [Neuronal Preparation on Coverslips with Glia Monolayer]
After dissection, place tubes containing SF1C for rinsing and triturating in the incubator until ready to triturate so that the pH stays between and , and the media is warm.
The media must be at the correct pH (indicated by the red color) or the neurons may die.
?. [Neuronal Preparation on Coverslips with Glia Monolayer]
TRITURATIONFrom this point forward, everything should be done in a sterile laminar hood with sterile technique.
?. [Neuronal Preparation on Coverslips with Glia Monolayer]
Remove chunks from enzyme vial using transfer pipette and place in 15 ml conical test tube.
IMPORTANT: Take great care to prevent bubble formation because bubbles are harmful to neurons. If bubbles occur, aspirate them off.
?. [Neuronal Preparation on Coverslips with Glia Monolayer]
Rinse chunks 2-3 times using about  warmed to . After adding SF1C for each wash, flick the tube so that the cells swirl around and shake off the papain. Allow tissue to settle each time and discard as much of the supernatant as possible without exposing cells to air.
[SF1C]
37 °C
?. [Neuronal Preparation on Coverslips with Glia Monolayer]
Triturate in  with large-bore tech-tips a couple of times (when done properly the medium becomes slightly opalescent with dissociated cells).
[room temperature SF1C]
?. [Neuronal Preparation on Coverslips with Glia Monolayer]
Repeat the trituration process up to three times with a medium-, then a small-bore tech-tip. The chunks should now be completely dissociated into individual cells.
?. [Neuronal Preparation on Coverslips with Glia Monolayer]
Centrifuge cells at .
Centrifuge: 1000 33, 5 min
?. [Neuronal Preparation on Coverslips with Glia Monolayer]
ATTACHMENTAfter , cells should have attached to the surface of the well. Remove rings from each well using forceps, which have sterile pipette tips at the end.
?. [Neuronal Preparation on Coverslips with Glia Monolayer]
Add   to each culture for a final concentration of 10 ng/ml GDNF (see media and solutions section how to prepare GDNF).
[GDNF (200 ng/ml)]
?. [NEXT DAY - Mitotic Inhibition]
The next day inhibit the growth of non-neural cells with 5-fluorodeoxyuridine (FDU). Dilute 1000X stock 1:10 by adding  to .
[stock]
[MEM]
?. [NEXT DAY - Mitotic Inhibition]
Media should be changed only when necessary (change of color) – aspirate half the media and add fresh preconditioned (pre-warmed at ).
37 °C
Try to avoid this step if possible as neuronal cells are very sensitive to the media exchange.
?. [NEXT DAY - Mitotic Inhibition]
Disturb cultures as little as possible, avoiding multiple observations (especially in the first 2 days after culturing).
?. [NEXT DAY - Mitotic Inhibition]
Process cells as needed for the individual experiment (ideally, between 5 days to 2 weeks old).
If necessary after fixation/immunostaining, the coverslips can be taken off the Petri dish (cut between the glass and Sylgard with a scalpel – careful not to break the glass!!!) and mounted on a glass slide. Alternatively, cells can be cultured on free-floating coverslips, which can be later removed from dishes for the experiments.
?. [Microglial Culture]
Obtain one or two rat or five mouse pups P1-P3.
Prepare astrocyte cultures in tissue culture flasks (225 cm2 for rat and 75 cm2 for mice cultures)
?. [Microglial Culture]
Anesthetize pups with an intraperitoneal injection of ketamine.
?. [Microglial Culture]
When pups begin to show sedation, put them  till hypothermic.
on ice
?. [Microglial Culture]
Rinse aluminum foil square, scissors and head with 70% EtOH.
?. [Microglial Culture]
Decapitate pup, allowing head to fall onto aluminum foil square and move to hood.
?. [Microglial Culture]
Remove the olfactory lobes and brain stem (with v-cut).
?. [Microglial Culture]
DIGESTIONPlace chunks in papain as with neuronal prep, without kynurenate (1 rat cortex/vial, or 5 mouse cortexes/vial). Chunks will be digested enough to triturate during the initial  to . We judge by the appearance of the chunks (like cotton candy) when they are ready
?. [Microglial Culture]
TRITURATION AND PLATINGWarm up sterile M10C-G to  for rinsing and trituration.
37 °C
?. [Microglial Culture]
Remove chunks from enzyme vial using transfer pipette and place in 15 ml conical test tube
?. [Microglial Culture]
Rinse chunks 3 times using , allowing them to settle each time and discard supernatant.
[warm M10C-G]
?. [Microglial Culture]
Using  triturate 5-10 times using just the regular pipette tip.
[warm M10CG]
?. [Microglial Culture]
Repeat trituration using the tech-tip with smaller size hole, until the chunks have all fallen apart.
?. [Microglial Culture]
Pellet the glial cells at .
Centrifuge: 1000 33, 5 min
?. [Microglial Culture]
Transfer cell suspension to tissue culture flask. Use big T-225 flask for rat culture and T-75 flask for mouse culture. The yield from two rat brains is ideal for one T-225 flask. And the yield from 5 mouse brains is ideal for one T-75 flask.
?. [Microglial Culture]
Allow cells to settle for at least , better leave them  in incubator.
?. [Microglial Culture]
RINSING AND FEEDINGFor astrocyte monolayer remove M10CG from flask and squirt ~ onto the base of upright tissue culture flask.
[cold MEM]
?. [Microglial Culture]
Aspirate off MEM and feed with cold M10C-G (30 ml per T-225 flask, or 15ml per T-75 flask), and leave ~2 weeks in the incubator.
DO NOT ADD FDU TO FLASKS!!!!
?. [Microglial Culture]
MICROGLIA ISOLATIONOnce the cell layers are stable and you start seeing rounded microglial cells on top of the layers (usually 10-14 d after the cortical culture is plated) and floating in the medium. You should harvest the floating microglia at this time.
?. [Microglial Culture]
Shake the flask with a back-and-forth motion several times to help lift the microglia off.
?. [Microglial Culture]
Collect the fluid in two 15 ml conical tube and centrifuge at  .
Centrifuge: 3000 33, 5 min
Note that the speed is higher than for regular cells centrifugation, which is normally 1,000rpm.
?. [Microglial Culture]
Remove supernate and GENTLY resuspend the cell pellet in  with regular 1ml pipette tip.
[warm conditioned M10CG]
?. [Microglial Culture]
Count cells on hematocytometer to determine appropriate volume for cells density @ 60,000 cells/well on neuronal cultures or 150, 000 cells/well 96 well plates for biochemical assays.
?. [Microglial Culture]
When plating on neuronal cultures place plastic ring around the well and plate cells in the ring. Remove ring after .
PLEASE BE SURE TO REMOVE NEURONAL MEDIA (SFIC) AND REPLACE WITH 2-3 DAY ASTROGLIAL CONDITIONED MEDIA BEFORE PLATING MICROGLIA.
?. [Neuronal Preparation on Coverslips with Glia Monolayer]
Make the initial cut through the entire brain caudal to the midbrain flexure (right line, Fig 1 in Guidelines section), and the second cut rostral to the flexure, including the caudal aspect of the hypothalamus.
?. [Neuronal Preparation on Coverslips with Glia Monolayer]
Remove any remaining cortex.
?. [Neuronal Preparation on Coverslips with Glia Monolayer]
Lay the resulting slice flat on the SYLGARD® with the ventral side facing right, and the dorsal side to the left and the caudal aspect up.
?. [Neuronal Preparation on Coverslips with Glia Monolayer]
Place the forceps through the aqueduct and push into the SYLGARD® to immobilize the slice.
?. [Neuronal Preparation on Coverslips with Glia Monolayer]
Cut the ventral edge of the slice along the top of the hypothalamus as indicated by the line in Figure in the Guidelines section.
?. [Neuronal Preparation on Coverslips with Glia Monolayer]
Cut into 1 mm3  segments and place all three segments into the vial with papain solution for ventral midbrain cultures. For SN cultures only use the two outer segments and for VTA only use the inner segment. Use both SN and VTA for combined VM cultures.
?. [Neuronal Preparation on Coverslips with Glia Monolayer]
Next cut approximately halfway between the new ventral edge of the slice and the aqueduct.
?. [Neuronal Preparation on Coverslips with Glia Monolayer]
Replace MEM with  .
[SF1C]
?. [Neuronal Preparation on Coverslips with Glia Monolayer]
Leave dishes with SF1C in the incubator . Alternatively, do this step on the day of the culture, using preconditioned media.
?. [Neuronal Preparation on Coverslips with Glia Monolayer]
Resuspend the pellet in 0.2-0.5 ml (for mouse cultures) or 1-2 ml (for rat cultures) of media, mix and count (see below).
?. [Neuronal Preparation on Coverslips with Glia Monolayer]
Add the appropriate volume to the well (into the slide ring) of the SF1C-containing dishes so that each well contains 80,000 cells (use a circular motion to spread the cells around). Again, the rings should ensure that the neurons settle into the well and do not float away or get washed away when moving the dishes.
?. [Neuronal Preparation on Coverslips with Glia Monolayer]
Dilute cell suspension until - of media contains 80,000 cells.
50 µl
100 µl
?. [NEXT DAY - Mitotic Inhibition]
Add to each dish (2 ml media).
[diluted FDU solution]
?. [Microglial Culture]
Gently remove brain into ice cold PBS filled Petri dish (with one Sylgard circle – should be covered with cold PBS!).
?. [Microglial Culture]
Hemi-sect brain and then peel off meninges.
?. [Microglial Culture]
Remove any torn areas to avoid DNA release.
?. [Microglial Culture]
Shell out cortex.
?. [Microglial Culture]
Cut into 1 mm3 chunks.
?. [Microglial Culture]
Gently allow the media to swirl around the bottom of the flask to remove unattached cells.
?. [Microglial Culture]
Shake additional ~ placing the flask on a shaker at moderate speed.
?. [Glia Preparation on Coverslip Petri Dishes]
Using (toothed) forceps hold head by the eyes (using left hand if you are right handed).
?. [Glia Preparation on Coverslip Petri Dishes]
Gently remove brain (using a small scoop shaped scalpel) and place into Petri dish with one SYLGARD® circle stuck onto the bottom and filled with ice cold sterile PBS (use enough PBS such that the brain lying on the SYLGARD® circle is submerged)
?. [Glia Preparation on Coverslip Petri Dishes]
Next, use a micro-scissor to cut around the circumference of the skull, and gently peel the skull off with forceps, being careful in case any tissue still connects it to the rest of the skull.
?. [Glia Preparation on Coverslip Petri Dishes]
Using the other hand and a curved or angled forceps, pinch the scalp just behind the eyes and pull back, tearing it off.
?. [Aclar Dishes for Electron Microscope Preparations]
Rinse 1/2: rinse in Nanopure dd H2O for .
?. [Aclar Dishes for Electron Microscope Preparations]
Rinse 2/2: rinse in Nanopure dd H2O for .
?. [Aclar Dishes for Electron Microscope Preparations]
Rinse 1/2: rinse in Nanopure dd H2O for .
?. [Aclar Dishes for Electron Microscope Preparations]
Rinse 2/2: rinse in Nanopure dd H2O for .
?. [Aclar Dishes for Electron Microscope Preparations]
Rinse 2/3: rinse in tissue culture water for .
?. [Aclar Dishes for Electron Microscope Preparations]
Rinse 3/3: rinse in tissue culture water for .
?. [Glia Preparation on Coverslip Petri Dishes]
Rinse 1/3: Rinse chunks using , allow them to settle and discard supernatant.
[M10C-G]
?. [Glia Preparation on Coverslip Petri Dishes]
Rinse 2/3: Rinse chunks using , allow them to settle and discard supernatant.
[M10C-G]
?. [Glia Preparation on Coverslip Petri Dishes]
Rinse 3/3: Rinse chunks using , allow them to settle and discard supernatant.
[M10C-G]
?. [Glia Preparation on Coverslip Petri Dishes]
Squirt  into each well, moving the pipette around in a circular motion to wash each well evenly.
[cold MEM]
?. [Glia Preparation on Coverslip Petri Dishes]
Aspirate off MEM.
?. [Neuronal Preparation on Coverslips with Glia Monolayer]
Add an HCl-cleaned, autoclaved micro-stir bar to the vial. Fill each tube with - (sterile filtered with 0.22 µm filter). Place in temperature controlled   water bath with magnetic stirrers.
5 mL
[freshly made papain solution]
37 °C
?. [Neuronal Preparation on Coverslips with Glia Monolayer]
Wash 1/2: wash with  .
[cold MEM]
?. [Neuronal Preparation on Coverslips with Glia Monolayer]
Wash 2/2: wash with  .
[cold MEM]
?. [Neuronal Preparation on Coverslips with Glia Monolayer]
Rinse 1/2: Rinse chunks 2-3 times using about  warmed to . After adding SF1C, flick the tube so that the cells swirl around and shake off the papain. Allow tissue to settle and discard as much of the supernatant as possible without exposing cells to air.
[SF1C]
37 °C
?. [Neuronal Preparation on Coverslips with Glia Monolayer]
Rinse 2/2: Rinse chunks 2-3 times using about  warmed to . After adding SF1C, flick the tube so that the cells swirl around and shake off the papain. Allow tissue to settle and discard as much of the supernatant as possible without exposing cells to air.
[SF1C]
37 °C
?. [Neuronal Preparation on Coverslips with Glia Monolayer]
Optional Rinse: Rinse chunks 2-3 times using about  warmed to . After adding SF1C, flick the tube so that the cells swirl around and shake off the papain. Allow tissue to settle and discard as much of the supernatant as possible without exposing cells to air.
[SF1C]
37 °C
?. [Microglial Culture]
Rinse 1/3: Rinse chunks using , allow them to settle and discard supernatant.
[M10C-G]
?. [Microglial Culture]
Rinse 2/3: Rinse chunks using , allow them to settle and discard supernatant.
[M10C-G]
?. [Microglial Culture]
Rinse 3/3: Rinse chunks using , allow them to settle and discard supernatant.
[M10C-G]
?. [Microglial Culture]
Resuspend pellet in .
[ M10C-G]