Begin by centrifuging the isolated brain cell suspension at 300G for eight minutes. Resuspend the pellet in 80 microliters of HBSS with 0.5%BSA solution. Add 20 microliters of non neuronal cell biotin antibody cocktail and incubate.
Wash the cells with two milliliters of HBSS with 0.5%BSA. Then resuspend the centrifuge pellet in 80 microliters of HBSS with 0.5%BSA. Now add 20 microliters of anti biotin microbeads, and pipette to mix thoroughly.
After cold incubation add 0.5 milliliters of HBSS BSA solution. Set up the stand before rinsing the column with 0.5 milliliters of HBSS with 0.5%BSA. Next place a 15 milliliter tube under the column and pass 0.5 milliliters of the cell suspension through it.
Perform three washes with 0.5 milliliters of HBSS with 0.5%BSA solution to capture residual neuronal cells. Remove the column from the magnet and place it inside a new 15 milliliter tube. Add one milliliter of HBSS with 0.5%BSA and use the plunger to collect magnetically labeled non neuronal cells.
After cell centrifugation, resuspend the cells in one milliliter of HBSS with 0.5%BSA solution. Count the cells on a neubauer counting chamber under a brightfield microscope. LepR positive neurons began to form neurites after 48 hours.
At DIV4 axonal extensions showed progress while dendritic processes began to appear. At DIV6 the neurons were sufficiently developed. Immunofluorescent studies showed that no glial cells or other non neuronal cells were observed.
The neuronal nature of the cells was confirmed by microtubule associated protein two staining. At DIV10 30%of LepR positive cells expressed POMC. Synaptic connectivity and functionality were observed in general cultures containing heterogeneous hypothalamic neuronal populations.
