May 11, 2026
Preparation for E13 embryonic mouse spinal cord cultures
- Enock Mararo1
- 1University of Glasgow
Protocol Citation: Enock Mararo 2026. Preparation for E13 embryonic mouse spinal cord cultures. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vz4zrrlx1/v1
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 09, 2026
Last Modified: May 11, 2026
Protocol Integer ID: 316674
Keywords: establishing primary spinal cord cell culture, primary spinal cord cell culture, preparation for e13 embryonic mouse, spinal cord cultures this protocol, spinal cord culture, tissue culture, e13 embryonic mouse, dissecting embryonic tissue, embryonic tissue, preparation step, dissection bench setup, preparation, gathering surgical instrument, surgical instrument, bench setup, tc setup, microscope, biosafety cabinet
Abstract
This protocol outlines the preparation steps for dissecting embryonic tissue and establishing primary spinal cord cell cultures. It is divided into two phases: dissection bench setup and tissue culture (TC) suite preparation. The bench setup covers gathering surgical instruments, the microscope, and cold reagents like Trypsin and HBSS. The TC setup covers preparing the biosafety cabinet, culture media, PLL-coated dishes, and Erythrosin B for cell counting.
Materials
- Large ice bucket with ice
- Dissecting microscope (booked in advance)
- Dissecting instruments and large scissors/forceps for cervical dislocation of dam
- Sterile 10 cm Petri-dish on ice, lid on
- 10x trypsin, on ice to thaw
- ~ 4 ml HBSS minus Ca2+ and Mg2+ (HBSS -/-), on ice
- 2 x sterile 35 mm Petri-dishes for dissecting embryos and removing meninges
- 7 ml bijou (e.g. Star Lab E1412-0710)
- Labelled 0.5 ml tubes
- 1 x 100 µl and 1 x 1000 µl pipettes and filter-top tips
- Sterile scalpel blade and blade handle
- Open cell culture hood, turn fan on and spray hood with 70% ethanol
- Pipettes, Pipettman, pipette tips, serological pipettes, 5 ml syringe and 1 each of 21 and 23 gauge needles
- ‘Plating media’ (PM) and ‘differentiation media plus insulin’ (DM+)
- DNAse on ice or in fridge to thaw
- 2 x 13 ml conical centrifuge tubes
- Haemocytometer and pink Erythrosin B
- 35 mm Petri-dishes coated with PLL in boric acid buffer
Prepare bench for dissection
Prepare a large ice bucket with ice to keep your reagents cold.
Clean the dissecting microscope (booked in advance) on the bench prior to use.
Put dissecting instruments and large scissors/forceps for cervical dislocation of dam on a bit of blue roll and sterilise with 70% methylated spirit (ethanol).
Place a sterile 10 cm Petri-dish on ice, lid on, on a small white tray next to the dissecting microscope.
Locate 10x trypsin in the TC freezer in room 343, in the labelled boxes. Place on ice alongside the petri dishes and bijou tubes.
Retrieve ~ 4 ml HBSS minus Ca2+ and Mg2+ (HBSS -/-) from the cold room and place on ice.
Prepare 2 x sterile 35 mm Petri-dishes for dissecting embryos and removing meninges.
Label a 7 ml bijou (e.g. Star Lab E1412-0710) with your initials on the lid and side. Place on ice.
Prepare labelled 0.5 ml tubes if the embryos/mother need to be genotyped.
Prepare 1 x 100 µl and 1 x 1000 µl pipettes and filter-top tips.
Prepare a sterile scalpel blade and blade handle.
Prepare cell culture suite (TC setup)
Open cell culture hood, turn fan on and spray hood with 70% ethanol. Set the hood up as normal.
Ensure you have pipettes, Pipettman, pipette tips, serological pipettes, 5 ml syringe and 1 each of 21 and 23 gauge needles ready in the hood.
Retrieve ‘Plating media’ (PM) and ‘differentiation media plus insulin’ (DM+) aliquots with your colleagues' initials from the fridge. Place in 50 ml Greiner tubes, at room temperature, upright in a rack.
Retrieve a minimum of 2 aliquots of DNAse from the TC freezer and place them on ice.
Label 2 x 13 ml conical centrifuge tubes with your initials and "Tube 1" and "Tube 2", plus a balance tube containing water and a rack to hold them upright.
Retrieve the pink Erythrosin B aliquots from the bottom drawer of the lab trolley for use with the haemocytometer. Trypan blue is no longer used for cell counting.
Retrieve 35 mm Petri-dishes coated with PLL in boric acid buffer from the cold room, located in the pink box on the cell culture shelf. Place them inside larger 10 or 25 cm Petri-dishes, labelled with your name, the date, and the experiment number.