Begin by centrifuging the murine-pure neuronal cell suspension at 300 G for eight minutes. Gently aspirate the supernatant and resuspend the pellet in 80 microliters of HBSS with 0.5 BSA solution. After adding the specific antibody, incubate the tubes at four degrees Celsius for 10 minutes.
Wash the excess antibody with HBSS-BSA solution and centrifuge. Add 20 microliters of antibiotin microbeads to the pellet, resuspended in HBSS-BSA. Incubate the tube at four degrees Celsius for 10 minutes.
Then, add 0.5 milliliters of HBSS with 0.5%BSA solution for every 10 to the seven cells. Rinse the column with 0.5 milliliters of HBSS with 0.5%BSA solution. Once the dripping stops, place a 15-milliliter tube beneath the column and pass 0.5 milliliters of cell suspension through it.
Collect the eluate containing nonspecific neuronal cells, then clean the column with three washes of 0.5 milliliters of HBSS with 0.5%BSA. Now remove the column from the magnet and place it in a new 15-milliliter tube. Add one milliliter of HBSS with 0.5%BSA and use the plunger to flush out the targeted cells.
After centrifugation and supernatant aspiration, resuspend the pellet in 0.5 milliliters of plating medium. Count the cells in a Neubauer counting chamber under a bright-field microscope. Plate the targeted cells as the positive control and non-specific cells as the negative control in the prepared 24-well plate.
Incubate at 37 degrees Celsius for 12 hours. 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 2 staining.
At DIV10, 30%of LepR-positive cells expressed POMC. Synaptic connectivity and functionality were observed in general cultures containing heterogeneous hypothalmic neuronal populations.
