Tetrazine-labeling of uAA-tagged proteins in dissociated hippocampal neurons
Day 0: Dissociation of rat hippocampal neurons
⚠ Dissociated hippocampal neurons from embryonic day 18 (E18) Sprague-Dawley rats embryos of both sexes were prepared in Banker-like cultures as previously described 15 with modifications. For a detailed protocol please refer to 15.
Dissociated neurons were plated at a density of 250,000 cells per 60 mm culture dish on 0.1 mg.mL-1 PLL pre-coated 1.5H, ⌀ 18 mm coverslips – 4 coverslips per 60 mm culture dish. Neuron cultures were maintained in Neurobasal Plus Medium supplemented with 0.5 mM GlutaMAX and 1X B-27™ Plus Supplement.
Hippocampal astrocytes feeder layers were prepared two weeks in advance from E18 embryos, plated between 20,000 to 40,000 cells per 60 mm culture dish and cultured in Minimum Essential Medium containing 4.5 g.L-1 glucose, 2 mM GlutaMAX and 10% heat-inactivated horse serum for 14 days.
Day 3:
- Add 2 uM of Cytosine β-D-arabinofuranoside to the 60 mm culture dish containing the hippocampal neurons.
Day 3-4: Lipofectamine 2000 transfection
- In a 12-MW plate, add 500 uL of Neurobasal media pre-equilibrated at 37ºC per well;
- Transfer the coverslips containing the neurons to be transfected from the 60 mm culture dish to the 12-MW plate and store it in the incubator;
- Lipofectamine 2000 transfection (for 4 coverslips, i.e., 1x 60 mm culture dish):
o Prepare and label two 1.5 mL eppendors, A and B
§ epp. A: Mix 0.417 ug of pTRE3G-BI PylRS/γ2 S44*, 0.417 μg uL of Tet3G/tRNAPyl, and 0.166 μg of eGFP in 100 μL of OptiMEM at RT.
§ epp. B: Add 4 μL of Lipofectamine 2000 into 100 μL of OptiMEM. Mix by gently tapping the Eppendorf with the fingers.
o Add DNA mix solution from epp. A dropwise to the epp. B. Mix by gently tapping the Eppendorf with the fingers;
o Incubate for 5 min at RT;
o Take the 12-MW plate containing the coverslips from the incubator and add 50 uL of the DNA-lipofectamine mix solution dropwise per coverslip. Homogenate the solution by gently tilting the plate;
o Incubate for 45 min at 37ºC;
o Remove the transfection media from 12-MW plate, and rinse the coverslips with 250 μL of Neurobasal media pre-equilibrated at 37ºC;
o Transfer the coverslips back to the respective 60 mm culture dishes.
Day 10-11:
- Add 2 mL of fresh Neurobasal media pre-equilibrated at 37ºC per 60 mm culture dish.
Day 15-17: uAA supplementation
- Prepare a 25 mM TCO*A working solution (for 4 coverslips): Add 30 μL of 1M HEPES to 10 μL of TCO*A stock solution (see reagents);
- Prepare a 50 mg/mL doxycycline working solution: Dilute 2 uL of doxycycline stock solution in 78 μL of cell media;
- In a 12-MW plate, add 1 mL per well of cell media from the transfected neurons-containing 60 mm culture;
- Add 10 uL of TCO*A working solution and 2 μL of doxycycline working solution per well of 12-MW plate containing 1 mL of cell media. Final concentration: 250 μM of TCO*A and 100 ng/mL of doxycycline solution;
- Transfer the converslips containing the transfected neurons to the 12-MW plate and return it to the incubator.
Day 16-18: Tetrazine-labeling (~18-22 h after TCO*A and doxycycline treatment)
- Prepare 10 mL of Tyrode’s solution containing 1% BSA. Equilibrate at 37ºC.
- Remove TCO*A- and doxycycline-containing cell media;
o Avoid doing more than 4 coverslips at a time.
- Rinse coverslips 3 times with ~1 mL of Tyrode’s solultion pre-warmed at 37ºC;
- Wash for 3 min with ~1 mL of 1%BSA-Tyrode’s sol pre-warmed at 37ºC;
- Prepare 0.5 μM of Pyr-Tet-ATTO643 solution (for 4 coverslips): Dilute 2 μL of 500 μM Pyr-Tet-ATTO643 (diluted in DMSO) in 2 mL of warm 1% BSA-Tyrode’s solution. Mix vigorously by pipetting;
- Add 250 uL of 0.5 μM of H-Tet-Cy5 solution per well and incubate for 10 min @37ºC;
- Rinse cells 4 times with ~1 mL of Tyrode’s sol pre-warmed @37ºC;
- Transfer the coverslips to a metal Ludin chamber and add 1 mL of ward Tyrode’s solution.
Image live neurons using a spinning disk confocal microscope or equivalent (Figure 1).
Tetrazine-labeling of uAA-tagged proteins in organotypic hippocampal slice cultures (OHSC)
Day 0: Culture of mouse hippocampal slices
⚠ Organotypic hippocampal slice cultures from postnatal day 5-7 from C57Bl6/J mice of both sexes were prepared as previously described 16 with modifications. For a detailed protocol please refer to 16.
Animals were anesthetized on ice and sacrificed by decapitation. Hippocampi were dissected out and placed in ice-cold carbonated dissection buffer (see reagents). Coronal slices (300 µm) were cut using a tissue chopper, collected and positioned on interface-style Millicell culture inserts in 6 well culture plates containing 1 mL of sterile OHSC media (see reagents) pre-equilibrated @35ºC. Brain slices were incubated @35 °C under 5% CO2 and the culture medium was changed from the bottom of each well every 2-3 days.
Day 11-13: single-cell electroporation
⚠ Single-cell electroporation of CA1 pyramidal neurons in OHSC was performed as previously described 17 with modifications. For a detailed protocol please refer to 17.
- Prepare glass micropipettes with resistance ~4-6 MΩ using a pipette puller;
- Prepare about 70 uL of DNA solution: Dilute 26 ng/uL of pTRE3G-BI PylRS/γ2 S44*, 26 ng/μL of Tet3G/tRNAPyl, and 13 ng/uL of eGFP in ~70 μL of K-gluconate based intracellular solution.
- Fill the glass micropipettes with ~6 μL of DNA solution (electroporation pipette). Mount the electroporation pipette in the micromanipulator;
- Fill the microscope recording chamber with ~2 mL warmed ACSF (see reagents). Transfer one slice from the 6-MW plate to the microscope recording chamber;
- Approach the selected cells with the electroporation pipette while applying positive pressure;
- After a loose seal formation apply 4 x 25 ms pulses at 1 Hz, -2.5V;
- Slowly retract the electroporation pipette and begin applying very light pressure when 2-5 μm away from the soma. Increase pressure at larger distances;
- Repeat the process. ⚠ Avoid keeping the slice for longer than 20 min;
- Return the slice to the 6-MW plate. Return the plate to the incubator.
Day 14-16: uAA supplementation
- Prepare a 25 mM TCO*A working solution (for 4 wells of a 6-MW plate): Add 30 μL of 1M HEPES to 10 μL of TCO*A stock solution;
- Prepare a 50 mg/mL doxycycline working solution: Dilute 2 μL of doxycycline stock solution in 78 μL of cell media;
- Add 10 μL of TCO*A working solution and 2 μL of doxycycline working solution per well of 6-MW plate containing 1 mL of OHSC media. Final concentration: 250 uM of TCO*A and 100 ng/mL of doxycycline solution.
Day 15-17: Tetrazine-labeling (~20-22 h after TCO*A and doxycycline treatment)
- Prepare 10 mL of ACSF containing 1% BSA. Equilibrate at 35ºC.
- Remove TCO*A- and doxycycline-containing OHSC media;
- Wash slices 3 times for 5 min with ~2 mL (1 mL outside and 1 mL within the culture inserts) of ACSF pre-warmed at 35ºC. Make sure the slices are submerged;
- Wash for 5 min with ~2 mL (1 mL outside and 1 mL within the culture inserts) of 1%BSA-ACSF pre-warmed at 35ºC. Make sure the slices are submerged;
- Prepare 1 μM of H-Tet-Cy5 solution (for 4 wells): Dilute 8 uL of 500 uM H-Tet-Cy5 (diluted in DMSO) in 4 mL of warm 1% BSA-ACSF. Mix vigorously by pipetting;
- Add 1 mL of 1 uM of H-Tet-Cy5 solution to the culture insert. Make sure the slices are submerged. Incubate for 10 min @35ºC;
- Wash slices 4 times for 5 min with ~2 mL (1 mL outside and 1 mL within the culture inserts) of ACSF pre-warmed at 35ºC. Make sure the slices are submerged;
- Remove ACSF and fix slices with 4% PFA (1 mL outside and 1 mL within the culture inserts) for 2 h @RT. Make sure the slices are submerged;
- Wash slices 3 times for 5 min with ~2 mL (1 mL outside and 1 mL within the culture inserts) of PBS;
- Block reactive aldehyde groups for 20 min with ~2 mL (1 mL outside and 1 mL within the culture inserts) of 200 mM NH4Cl @RT;
- Rinse slices with ~2 mL (1 mL outside and 1 mL within the culture inserts) of PBS;
- Transfer the slices to a glass microscope slide. Mount slices in Fluoromount-G Mounting medium. Cover slices with a slide coverslip. Let slices cure for 48 h at RT protected from light.
Image slices using a point scanning confocal microscopy or equivalent (Figure 2).