Oct 07, 2022
Preparation of organotypic cerebellar cultures
- Laura Lossi1,
- Adalberto Merighi1
- 1Department of Veterinary Sciences - University of Turin
Protocol Citation: Laura Lossi, Adalberto Merighi 2022. Preparation of organotypic cerebellar cultures. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr67bbvmk/v1
Manuscript citation:
Alasia, S.; Merighi, A.; Lossi, L. 2015; Transfection Techniques and Combined Immunocytochemistry in Cell Cultures and Organotypic Slices. In Immunocytochemistry and Related Techniques, Merighi, A.; Lossi, L., Eds. Springer New York: Vol. 101, pp 329-355.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 27, 2022
Last Modified: October 07, 2022
Protocol Integer ID: 63334
Keywords: organotypic cultures, brain, cerebellum, neural development, immunocytochemistry, live imaging
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Abstract
This protocol describes the basics to prepare organotypic cerebellar cultures using the membrane interface method.
Guidelines
All procedures must be carried out under sterile conditions.
Materials
Pentobarbital sodiumSigma AldrichCatalog #Y0002194
D-( )-GlucoseSigma-aldrichCatalog #G8270 L-Ascorbic acidSigma – AldrichCatalog #A92902 Pyruvic acidSigma-aldrichCatalog #107360 N-Methyl-D-glucamineSigma-aldrichCatalog #M2004 Sodium bicarbonateSigma AldrichCatalog #S5761 Potassium chlorideSigma AldrichCatalog #P3911 Sodium phosphate monobasicSigma – AldrichCatalog #S0751 Calcium chlorideSigma – AldrichCatalog #C1016 Magnesium chlorideSigma – AldrichCatalog #M8266 Basal Medium EagleSigma – AldrichCatalog #B9638 Horse serumSigma – AldrichCatalog #H1138 Hanks′ Balanced Salt solutionSigma – AldrichCatalog #H6648 L-Glutamine solutionSigma – AldrichCatalog #G7513 Antibiotic Antimycotic Solution (100×) StabilizedSigma – AldrichCatalog #A5955
Equipment
McIlwain Tissue Chopper with Petri Dish Modification
NAME
Tissue chopper
TYPE
Campden Instruments
BRAND
Model TC752-PD
SKU
Equipment
new equipment
NAME
Stereo microscope EZ4, Leica
BRAND
10447197
SKU
Leica EZ4 educational stereomicroscope.
SPECIFICATIONS
Equipment
Certomat CS-18
NAME
CO2 incubator
TYPE
Sartorius
BRAND
BBI-8863385
SKU
Dissecting tools: universal scissors, fine scissors straight and curved, Adson forceps, student anatomical standard pattern forceps, Dumont #7 forceps, gross anatomy blade (#20) and handle (#4), straight and curved spatulas, razor blades.
Safety warnings
Be sure to turn off the UV light when using the laminar flow hoods.
Before start
Be sure to have all your tool and solutions ready. Working areas must be clean and sterilized with UV ligh or 70% ethanol.
Preparation of solutions/culture medium
Preparation of solutions/culture medium
1h 45m
1h 45m
Solutions
Stock solutions: 1 Molarity (M) CaCl2; 1 Molarity (M) MgCl2;
5 % volume pentobarbital sodium in ddH20
30m
Cutting solution composition: 130 millimolar (mM) n-methyl-D-glucamine Cl (NMDG); 24 millimolar (mM) NaHCO3; 3.5 millimolar (mM) KCl; 1.25 millimolar (mM) NaH2PO4; 0.5 millimolar (mM) CaCl2; 5 millimolar (mM) MgCl2; 10 millimolar (mM) D-(+)-glucose; 1 mg/mL ascorbic acid; 2 mg/mL pyruvic acid.
Cutting solution preparation: To make 1 L , pour 850 mL double-distilled water into a volumetric flask. Add 25.38 g NMDG, 2.017 g NaHCO3, 261 mg KCl, 172 mg NaH2PO4, 1.80 g D-(+)-glucose, 1 g ascorbic acid, 2 g pyruvic acid. After complete dissolution SLOWLY add 5 mL MgCl2 stock solution and 500 µL CaCl2 stock solution.7.2 to 7.4 with HCl.
Note
The addition of MgCl2 and CaCl2 is a critical step. If MgCl2 and CaCl2 are added too quickly they precipitate making the solution cloudy. In this case, it must be discharged.
Steril filter and store at 4 °C . The solution is stable for several months. Discharge if it becomes turbid.
1h
Culture medium
Culture medium composition: 50% Basal medium Eagle (BME), 25% horse serum; 25% Hank's balanced salt solution (HBSS); 0.5 % D-(+)-glucose; 0.5% L-glutamine (200 mM solution); 1% antibiotic antimycotic solution (100 x)
Culture medium preparation: Work under a laminar flow hood and use sterile glassware/plasticware. In a 100 mL cylinder add the components in the order indicated above. For 50 mL: 25 mL BME, 12.5 mL horse serum; 12.5 mL HBSS; 250 µL D-(+)-glucose; 250 µL L-glutamine; 500 µL antibiotic antimycotic solution. Transfer in a glass bottle and protect from light with aluminum foil. Store at 4 °C . Medium is stable for at least six months. Discharge if color changes and/or it becomes turbid.
15m
Tissue sampling
Tissue sampling
30m
30m
Have ready the following: ice-cooled cutting solution; 50 mL sterile glass or plastic becker; 150 mm diameter sterile glass or plastic Petri dishes; sterile dissection/slice handling tools; sodium pentobarbital stock solution (Room temperature ); 500 µL disposable insulin syringes; sterile razor blades; sterile glass/disposable Pasteur pipettes; sterile filter paper dishes.
Note
Dissection of the brain and separation of individual slices after cutting (see Slice seeding below) should be carried out under sterile conditions as far as possible. If it is not possible to place the stereomicroscope under the laminar flow hood, dissection should be carried out under a simple plastic box opened in the front. The entire dissecting area should be clean and wiped off with 70 % volume ethanol.
Euthanize mice at the required post-natal age with an overdose of intraperitoneal sodium pentobarbital (60 mg ⁄ 100 g body weight).
5m
Quickly remove the brain from the skull while the head is kept submerged in the ice-cooled cutting solution and isolate the cerebellum under the stereomicroscope.
Note
During the production of slices, all procedures must be carried out in an ice-cold cutting solution.
To keep the temperature a few degrees above °C during the dissection, prepare some blocks of frozen cutting solution to be added to the 4 °C chilled solution contained in the Petri dish used to dissect the brain.
10m
Before cutting, completely remove the meninges with a pair of N.7 Dupont forceps. Place the cerebellum on the stage of the tissue chopper within a drop of the ice-cooled cutting solution. Operate the chopper and cut 350 μm-thick parasagittal slices. Once terminated slicing, collect slices with a curved spatula (they are usually stuck together) and place them in a sterile 50-mm Petri filled with the ice-cooled solution. Store at 4 °C until ready to separate slices. Separate individual slices under the stereomicroscope with a spatula and a needle, trying not to damage the tissue. During the entire procedure, slices must be submerged in the ice-cooled cutting solution. Discharge damaged slices.
Note
If the cerebellum is not submerged by an excess of cutting solution, cutting with the chopper is easier. Set section thickness to any value between 200 µm - 400 µm after wiping out the solution with a piece of filter paper. Other cutting parameters, such as blade force, must be adjusted based on the type of chopper in use.
Use a spatula with curved edges to collect slices and transfer them from the cutting stage of the chopper to the Petri dish.
15m
Slice seeding
Slice seeding
1h
1h
Before starting to seed slices onto the Millicell inserts bring the culture medium at Room temperature and fill some sterile 35-mm plastic Petri dishes with 1.1 mL medium. Work under sterile conditions.
Note
The number of dishes required depends on the number of recovered slices. In general, working with the mouse post-natal cerebellum each slice is about 5 mm2. Therefore, one can easily plate 5-6 slices/insert. Working with older animals or on larger areas of the brain, i.e. the cerebral cortex allows plating a maximum of (roughly) 3 slices/insert.
Collect slices one by one and carefully lift them onto the dry Millicell membrane. Once the required number of slices has been plated in the insert, place it inside a 35-mm Petri dish containing the required amount of medium. Be careful to avoid air bubbles forming between the insert membrane and the medium, i.e. check that the membrane's lower surface is completely wet.
1h
Incubate at 34 °C in 5 % volume CO2 for up to 30 days in vitro (DIV). Medium has to be changed twice a week. Allow slices to equilibrate to the in vitro conditions for at least 4-6 DIV before treatments (if applicable).
Note
Slices obtained from cerebellum (and other CNS areas) survive better at temperatures below 37 °C , hence the temperature settings of the incubator are important for culture survival.
However, it should be noted that the neuroprotective effect of mild hypothermia on cultured neurons may obscure the action of certain apoptotic inductors if one is interested in the study of cell death.