CSF Collection from Mice with Leptomeningeal Disease for Subsequent Clone Expansion

To begin, identify the female immunodeficient NSG mice, having leptomeningeal disease, or LMD. Place an anesthetized mouse on the surgical table. Position the nose of the mouse in a modified L-shaped cone of a stereotactic apparatus, ensuring that the nostrils stay unobstructed.

Pulling the skin gently forward across the ventral surfaces of both pinnae, then fix it to the nose cone with tape. Guide the surgical scissor tips downward from the pinnae across the occipital bone. Create a small midline incision measuring five to seven millimeters just above the palpated concavity.

With a pair of blunt tipped forceps having one to two millimeter tips, gently apply pressure on the cisterna magna. Introduce the tips in a closed position and open them while exerting downward pressure on the dura. Continue to perform blunt dissection until the dural membrane is clearly discernible and the associated blood vessels are visible within the exposed area.

Now keep the forceps open to retract the surrounding musculature. Then attach a 27 to 29 gauge needle to a one milliliter syringe. Insert the syringe beneath the dura to visualize the bevel.

Gradually retract the syringe plunger to collect as much cerebrospinal fluid as possible. Transfer the fluid into a microcentrifuge tube. Immediately place it on ice.

Centrifuge the sample at 257 g for five minutes at four degrees Celsius. After aspirating the supernatant, pipette 500 microliters of sterile PBS to the cell pellet. Centrifuge the pellet at 257 g for five minutes at room temperature.

Discard the supernatant, then transfer the pellet to a 96-well plate containing HMC conditioned media.