To begin, place the previously isolated brain from the euthanized mouse on a Petri dish filled with ice. Position the brain upside down, such that the cortex is ventral, and the hypothalamus is visible dorsally on the superior surface. Then, dissect the hypothalamus using curved forceps.
Collect the pieces of the hypothalamus in a dissociation or C-tube previously filled with 1.9 milliliters of buffer Z.Next, prepare 30 microliters of enzyme mix 2. In each C-tube, add 50 microliters of the enzyme contained in mix 1, and 30 microliters of enzyme mix 2. Place the C-tube containing hypothalamus pieces upside down in the dissociation with the heating brackets and run the adult brain dissociation kit program tube.
In the meantime, place a 70 micron strainer on the top of a 15 milliliter snap tube. Wet the strainer with two milliliters of DPBS. After dissociation, empty the C-tube onto the strainer.
Scratch the strainer with a 200 microliter tip to reduce cell loss by increasing filtration and reducing the adherence of homogenate on the filter. Wash the C-tube with five milliliters of DPBS and pour the suspension onto the strainer. Scratch the strainer with a 200 microliter tip.
Centrifuge the tissue homogenate and aspirate the supernatant completely, before resuspending the pellet with an appropriate volume of cold DPBS. Immediately mix the cell suspension with 450 microliters of density gradient reagent, then, dropwise, gently overlay two milliliters of cold DPBS onto the cell suspension. Centrifuge the suspension with slow acceleration and slow break.
From the three phases, carefully aspirate and discard the two top phases completely. Fill the tube with 1800 microliters of cold DPBS and gently invert three times. Now, centrifuge the mixture with full acceleration and full break.
Aspirate the supernatant and resuspend the pellet in two milliliters of DPBS, containing 0.5%BSA. Centrifuge the cell suspension and resuspend the pellet in 90 microliters of DPBS, containing BSA. Add 10 microliters of the CD11b microbead mixture, and mix well with the pipette.
Incubate the tube for 15 minutes at two to eight degrees Celsius in the refrigerator. Add one milliliter of DPBS, containing 0.5%BSA, and centrifuge the mixture. After aspirating the supernatant, resuspend the pellet in 500 microliters of DPBS, containing 0.5%BSA.
Launch the parcel two program on the separator. Place the small columns in the magnetic field. Rinse the columns with 500 microliters of DPBS, containing 0.5%BSA.
To collect the negative fraction of cells, place a collection plate under the columns and apply the cell suspension in the column. Wash the column with 500 microliters of DPBS, containing 0.5%BSA, and wait for the buffer to pass through the column. Transfer the total effluent of CD11b negative fraction from the wells of the collection plate to 15 milliliter tubes.
Next, remove the column from the separator. Place the column on a five milliliter tube and pipette one milliliter of DPBS, containing BSA, into the column. Immediately, apply a plunger to flush out the magnetically labeled cell fraction.
Count the CD11b positive cells on a hemocytometer, then centrifuge the cell suspension and discard the supernatant.
