Protocols

200 nm m-Torquer was synthesized by chemical assembly of magnetic nanoparticles (MNPs) onto a 200 nm polystyrene supports, following a previously reported method with minor modifications.

1. Octahedral zinc doped iron oxide nanoparticles (MNPs) were synthesized by the thermal decomposition method.(2) 1.65 mmol tris(2,4-pentanedionato)iron(III) (TCI), 1.65 mmol zinc 2,4-pentanedionate monohydrate (Alfa Aesar), 2.2 ml oleic acid (Sigma-Aldrich) and 10 ml benzyl ether (Sigma-Aldrich) were mixed all in a three-neck round-bottom flask. 

2. The mixture was heated to 290°C for 50 minutes and held at for an hour. 

3. After removing the heat source, the reaction flask was allowed to cool to room temperature. 

4. The product was washed several times via centrifugation using isopropanol and toluene. 

5. The resulting precipitate is suspended and stored in toluene.

6. By encapsulating MNPs in amine-functionalized silica, amine-functionalized MNPs were prepared. Azide functional groups were introduced by reacting 50 µmol of azido-dPEG12-TFP ester (Quantabio) with 1 nmol of amine-functionalized MNPs in DMSO shaking 8 hours at room temperature. 

7. The resulting product was purified via centrifugation and resuspended in DMSO.

8. DBCO-functionalized spherical polystyrene supports were prepared by reacting 2.5 nmol of DBCO-PEG5-TFP ester (Click Chemistry Tools) with 1 nmol of amine-functionalized polystyrene microspheres (0.2 µm, Polyscience) in DMSO shaking 8 hours at room temperature. 

9. The product was purified through centrifugation and resuspended in DMSO.

10. Subsequently, azide-functionalized MNPs and DBCO-functionalized spherical polystyrene supports were conjugated at a 2000:1 molar ratio in DMSO. 

11. After 8 hours, unconjugated excess MNPs were removed by centrifugation. 

12. For further functionalization, carboxylate groups were introduced by using DBCO-PEG5-COOH (Click Chemistry Tools) and succinic anhydride (Sigma Aldrich), each at a final concentration of 1 mg·mL-1. 

13. The resulting carboxylated m-Torquer was purified via centrifugation and suspended in a 1 mM phosphate buffer (pH 7.2).

14. To attached Myc antibody on the m-Torquer surface, 10 μg protein A (Sigma Aldrich) was first conjugated onto 1 mg carboxylated m-Torquer using EDC/NHS coupling and then purified it through centrifugation. 

15. 1 μg Myc antibody (Sigma Aldrich) was conjugated onto protein A conjugated m-torquer. 

16. The resulting m-Torquer with Myc antibody conjugation was purified through centrifugation and resuspended in a 1 mM phosphate buffer at a final concentration of 50 mg·mL-1, adjusting the pH to 7.2.

1. Anesthetize mouse by injecting a mixture of ketamine (100mg/kg) and xylazine (25mg/kg) (I.P.) using a 1.0 ml syringe with 33 gauge needle.

2. Once the mouse is anesthetized, take a shaver or clipper to trim fur on the ventral neck area. 

3. Remove excess fur away from the surgical area.

4. Lay the mouse on a heating pad (36-37°C) in a supine position.

5. Sterilize incision area on the mouse using betadine (povidine iodine) 

6. To affix the head of the mouse on the stereotactic surgical instrument (Model 940 Small Animal Stereotaxic Instrument with Digital Display Console, David Kopf Instruments, Tujunga, CA, USA), place the upper incisor teeth of the mouse into the standard bite bar 

7. Tighten the bar clamp just enough so the head is secure 

8. To prevent cervical rotation of the mouse's head, secure the ear bars behind the zygomatic arch. 

9. Using vannas straight micro scissors, incise down the midline of the head

10. Retract the skin on both sides using straight fine forceps 

11. Locate the bregma and set stereotaxic x (medial lateral axis), y (anteroposterior axis), z (dorsoventral axis) coordinates to 0,0,0 as an origin reference point 

12. Set x,y stereotaxic coordinates to injection area 

13. Drill the skull directly on the injection area x,y stereotaxic coordinates 

14. Fill the WPI Nanofil 10ul syringe (UMP3; WPI, Sarasota, FL, USA) with a 33 gauge needle and set z stereotaxic coordinate slowly to inject mTorquer/Piezo1 virus at 0.3ul/min (the same with magnetic nanoparticles) 

15. After injection, leave the needle in place for 5 minutes 

16. Gently withdraw the needle from the brain by increasing the z stereotaxic coordinate

17. Suture the incision

18. Return the mouse to a clean cage and monitor for 3 days

Preparations:

• 1x DPBS 20ml

• 4% PFA 40ml - 20ml for perfusion, 20ml post-fixation

1. Anesthize the mouse with ketamine/xylazine (i.p. injection)

2. Lateral Incision to open chest cavity

3. Diaphragm incision using scissors

4. Using a scissor clamp, lift the leather up slightly to secure it

5. Find the right atrium of the heart (the darkest part) and make a small hole with scissors

6. Insert a 25G needle into the left ventricle through the apex of the heart. Be careful not to puncture through the septum

   1. 1. Perfusion with 20ml PBS

      2. Check for whitening of the fingers, toes, liver and tip of the nose

7. Switch to 4% PFA 20ml and perfuse

8. Collect the brain

Mouse Cardiac perfusion fixation

1. Anesthetize the mouse with ketamine and xylazine solution (100 mg.kg^-1) and 10mg.kg-1 of xylazine)

2. Make lateral incision slightly under the xiphiod to open chest cavity to expose the diaphragm and liver

3. Make incision in the diaphgram to expose the lungs and the heart

4. Find the right atrium of the heart (the darkest chamber) and make a small incision with scissors

5. Insert 25G infusion needle through the left ventricle
   (NOTE: Make sure the needle doesn't go through the septum to avoid the solution flowing to the pulmonary system)

6. Connect the infusion needle 20ml syringe filled with 1x DPBS

7. Start the perfusion with DPBS. 

8. Once the blood is drained out, connect the infusion needle 20ml with 4% PFA and perfuse.