Developing a Neuron and Macrophage Coculture In Vitro
Transcrição
Before setting up the culture, pre-coat a 6-well plate with poly-D-lysine and laminin. Next, incubate the 6-well plate with 0.01 poly-D-lysine at 37 degrees Celsius for two hours or at 4 degrees Celsius overnight. After that, wash the plate twice with distilled water.
Then, incubate the plate with laminin solution at a concentration of 3 micrograms per milliliter for two hours at room temperature. Following this, wash the plate twice with distilled water, and dry the plate at room temperature for, at least, one hour.
Now, incise the skin overlying the vertebral column of a euthanized mouse with a surgical blade, and dissect the paravertebral muscles bilaterally to expose the vertebral bones. Remove the vertebral bones meticulously using a narrow-tipped surgical rondure until the DRG are fully exposed.
Under a dissecting microscope, remove the DRG bilaterally from S1 all the way up to the C1 level using iridectomy scissors and fine-tipped forceps. Transfer the DRG to a 1.5-milliliter Eppendorf tube using a blue pipette tip with a cut-off end.
Then, remove DMEM after a quick spin for several seconds using a mini-centrifuge. Add 1 milliliter of DMEM containing 125 units per milliliter type-11 collagenase and incubate the tube for 90 minutes with a gentle rotation using a twist or shaker at 37 degrees Celsius.
Afterward, discard collagenase-containing DMEM and add 1 milliliter of fresh DMEM. Transfer the DRG to a 15-milliliter conical tube using a blue pipette tip with a cut-off end. Then, pipette up and down gently, at least, 15 times to make a homogeneous cell suspension.
Centrifuge the tube at 239 g for three minutes and carefully discard the supernatant with floating debris. Add 1 milliliter of neurobasal medium supplemented with B27 and re-suspend the cell pellet by gently pipetting up and down five to 10 times.
Next, pass the cell suspension through a 70-micrometer cell strainer overlaid on top of a 50-milliliter conical tube. Then, plate all the collected DRG neurons onto two wells of the 6-well plate.
To prepare primary peritoneal macrophages from an adult mouse, incise the abdominal skin delicately to expose the peritoneum and avoid cutting the peritoneum to prevent leakage of lavage fluid.
Next, puncture the peritoneum using a syringe with a 22-gauge needle and inject 10 milliliters of ice-cold PBS into the peritoneal cavity. Gently massage the peritoneum for one to two minutes. Then, pull out the needle, and squeeze out the PBS through the needle puncture site, and collect the lavage fluid in a 50-milliliter conical tube.
Afterward, centrifuge the lavage fluid at 239 g for 10 minutes at 4 degrees Celsius to pellet the cellular components. Subsequently, re-suspend the pellet with 3 milliliters of the red blood cell lysis buffer for three minutes at room temperature.
Then, centrifuge the cell suspension again at 239 g for 10 minutes at 4 degrees Celsius. Re-suspend the pelleted cells in 1 milliliter of neurobasal B27 medium. Plate half of all collected macrophages on a cell culture insert with an effective area of 4.2 centimeters squared, which is placed on top of the well of dissociated DRG.
Four hours after the neuron macrophage have been co-cultured, add 2 microliters of 100-micromolar db-cyclic AMP solution to the neuron macrophage co-cultures. After 24 hours, fill an empty well with 1 milliliter of macrophage culture medium in the same 6-well plate.
Transfer the cell culture insert in the neuron macrophage co-culture to the empty well with macrophage culture medium.
