Hi Ilyes Nedjar,

Once you co culture neurons with the astrocytes , it is difficult to separate the neurons from the astrocytes. As you are planning to do the flow cytometry

you definitely can use dopamine neurons specific markers along with you marker of interest. There are several well-characterized dopamine markers that can aid your research such as Tyrosine hydroxylase (TH). So that You will more accurate results.

Hope that helps.

Masuma

You don't need get rid of glial cells in order to detect neuronal MHC-I.

This gene is considered to be expressed in all nucleated cells and so both glia and neurons will express it.

Hello, Ilyes!

Please You look at this article:

Article An Easy-to-Implement Protocol for Preparing Postnatal Ventra...

Dissection of the Ventral Midbrain (VM) for Neuronal Cultures – 1 h, See Figure ​Figure33

∗This section describes how to dissect the ventral midbrain from postnatal rodent brain. The VM is isolated using a slice cutting approach and can be separated into VTA and SN. Figure ​Figure33 gives a schematic overview of individual steps.

• simple • Put the brain onto the Sylgard pad with the ventral side facing up to see the midbrain flexure.
• simple • Hold the brain by inserting the fine forceps No. 5A into both hemispheres.
• simple • Gently remove the meninges over the hindbrain using the fine forceps No. 5.
• simple • Make a vertical cut with the scalpel at the level of the hindbrain. see Figure ​Figure3A3A, dotted line a.
• simple • Then make another vertical cut cranial to the midbrain flexure – see Figure ​Figure3A3A, dotted line b.
• simple • You will have an approximately 1 mm thick coronal brain slice of the mesencephalon. The IV ventricle should be visible on the caudal side of the slice.
• simple • Put the fine forceps No. 5A into the ventricle to hold the slice – Figure ​Figure3B3B.
• simple • Make a vertical cut between midbrain and hindbrain – see Figure ​Figure3B3B, dotted line c.
• simple • Then make another cut half-way between the 1st cut and the ventricle – see Figure ​Figure3B3B, dotted line d.

CRITICAL STEP! Sometimes there can be some meninges left. Make sure to remove them.

• simple • Divide the VM into SN and VTA by cutting 1/3, 1/3, 1/3. See dotted lines in Figure ​Figure3C3C.
• simple • Put tissue chunks in a 1.5 mL Eppendorf tubes containing cooled Hibernate A® minus Calcium, using a transfer pipette, put back on ice.
• simple • Repeat all steps with the next brain.
• simple • Pause point – tissue can be kept for about an hour in Hibernate A® minus Calcium before papain digestion is started.

Papain Digestion of VM Tissue – 30 min

∗The dissociation of VM tissue is done using a mild papain digestion. For dissociation, a standard 1000 μL pipet tip coated with FBS is used. The papain can be prepared before starting the dissection. Filtered solution can be kept at 37°C for up to 3 h, otherwise store at 4°C.

• simple • Remove Hibernate A® minus Calcium from the tube with the tissue chunks.
• simple • Add 1 mL of the activated and filtered papain solution.
• simple • Incubate for 10 min in the incubator at 37°C.
• simple • In the meantime, prepare the washing solution – add 20 μl of the DNase stock solution to 2 mL Hibernate A® minus Calcium, prepare twice if you do SN and VTA separately.
• simple • After 10 min take your cells from the incubator and remove the papain solution, rinse 4x with 500 μL of washing solution each time.
• simple • Add 1 mL of Hibernate A® minus Calcium.
• simple • Triturate with a FBS-coated 1000 μL pipet tip.
• simple • About 30–40 trituration steps in total should be sufficient to reach a single cell suspension, let tissue chunks settle after every 10 steps.

TIP! Use a FBS coated pipet tip. For this, pipette up and down several times in a small volume of FBS before triturating papain-digested tissue chunks.

OptiPrep Purification and Plating of VM Cells – 1 h

∗The OptiPrep purification is a critical step to obtain healthy neuron cultures. Neurons can survive without this step but cell debris can interfere with neuronal sprouting and development.

• simple • Put 1 mL of cell suspension in a 15 mL Falcon tube and add 3 mL of Hibernate A® minus Calcium.
• simple • Use the long Pasteur pipet to add a sublayer of 1–1.5 mL of the 6.2% OptiPrep solution.CRITICAL STEP! Make sure to obtain two separate layers. Take up the OptiPrep solution with a long Pasteur pipet, carefully go to the bottom of the Falcon tube and slowly release the OptiPrep solution.
• simple • Centrifuge for 15 min at 500 g, do not use the brake to avoid disturbing the layers.
• simple • After centrifugation remove about 3 mL from the top containing cell debris.
• simple • Collect cells by harvesting the turbid band just in between the two layers, put these 1–2 mL in a new 15 mL Falcon tube.
• simple • Add the same volume of Hibernate A® minus Calcium to your cells and centrifuge for 3 min at 500 g, no brake.
• simple • Re-suspend your pellet in 1 mL Hibernate A® minus Calcium.
• simple • Count your cells by loading 9 μL of the cell suspension onto the Hemocytometer.
• simple • According to the experimental setup plate 50,000 to 100,000 cells in the middle of each MatTek dish with the pre-established glia feeder layers and the pre-conditioned Postnatal neuron medium, let cells settle for 10–15 min.
• simple • Add 1 μL of GDNF stock solution to each dish with 2 mL media (10 ng/mL) (Burke et al., 1998).
• simple • The next day add 2 μL of the cytarabine stock solution to 2 mL media to avoid glia growth.

TIP! Slide rings can be used to keep the cells in the middle of the dish. Here, cell numbers should be reduced. Rings can be reused, wash with plenty of water, spray with ethanol and autoclave again.

• simple • Put one of the autoclaved slide rings in the middle of each MatTek dish with the pre-established glia feeder layers and the pre-conditioned Postnatal neuron medium.
• simple • Plate your cells in the middle of the slide ring.
• simple • Remove the slide rings the next day. Use forceps covered by sterile non-filter pipet tips to avoid contamination.The cells can be used at DIV 7-14, but also maintenance up to DIV 48 is possible.

TIP! Try to keep your cells without media change, they are healthier with conditioned media. If you must change media, keep half of the old media and add half of fresh preconditioned media.

Article Isolation and Culture of Hippocampal Neurons from Prenatal Mice