General Protocol for Tissue Culture of N2A Cells General TC Notes: 1.

Wipe down inside of Tissue Culture hood with 70% EtOH 2. Ensure that all media is warmed to 37C before using 3. Before moving any items under hood, dry with a paper towel, spray down with 70% EtOH, and dry again 4. Spray down gloved hands with 70% EtOH before working under hood 5. All incubations, unless otherwise noted, occur in a humidified incubator at 37C, 5% CO2 6. Always note passage number on plates. 7. Change media at least every 72 hours. For 10-cm dishes: To change media: Aspirate media and add 12mL of complete media (One part Eagles Minimum Essential Media, one part F12 media, 10% FBS, 1% pen/strep). Return plate to incubator To split cells: 1. 2. 3. 4. Aspirate old media Add 2mL Trypsin-EDTA solution directly to cells, incubate @ 37C for 3 minutes Add 10mL complete media to cell/trypsin solution and pipette up and down to resuspend Distribute cells to new 10-cm dishes as needed, then add complete media to 12mL. a. For a typical 1:2 split, distribute 6mL cell stock each to two new plates, then add 6mL of complete media to each plate. 5. Return plate to incubator For TC Flasks: To split cells: 1. Aspirate old media 2. Add 2mL Trypsin-EDTA solution directly to cells, incubate @ 37C for 2 minutes 3. Add 8mL DMEM (+ appropriate antibiotics)to dissociated cells. Pipette up and down to resuspend 4. Distribute cells to a new flask as needed, then add complete media to 12mL: a. For a 1:20 split, add 18mL of media to a flask, and 500 L of the cell stock To plate into 12-well plates: 1. From the cell stock generated above, mix 1mL of cell stock and 11mL of DMEM in a 15-mL falcon tube 2. Distribute 1mL of cells to each well of a 12-well plate 3. Cells should be ready to transfect the next day

General Protocol for N2A Lysis and Western Blotting of Lysates Cell Lysis: 1. Combine 1.6 mL of 1x SDS lysis buffer, 400L -mercaptoethanol, and 20L of protease inhibitor in a 15mL falcon tube 2. After allowing appropriate time for expression in transfected cells (usually 24 hours), Aspirate media from each well of a 12-well plate 3. Add 150L of 1x SDS lysis buffer to each well. Scrape and collect in microcentrifuge tubes. 4. Boil all samples at 100C for 15 minutes 5. During procedures, keep tubes on ice. Store at -80C. BCA Assay 1. 2. 3. 4. 5. 6. Make up albinum standards, diluted in M-PER/protease inhibitor according to provided protocol Pipette 25L of standards in technical duplicates into a 96-well clear assay plate Pipette 25L of samples in technical duplicates into the assay plate. Cover plate and incubate 30 minutes at 32C Uncover plate and load into a 96-well plate reader. Read plate at 260nm Calculate necessary volume for 1g of total protein

SDS-PAGE 1. 2. 3. 4. Make up MES buffer from 20x MES solution in milliQ H2O Load a 4-12% bis-tris gels into a vertical electrophoresis apparatus. Fill with MES buffer. In first lane, load 5L of benchmark-prestained protein ladder. Load 5L of each lysate solution into each well in collated duplicate (one for -tau, one for actin) 5. Run gel at 200V for 35 minutes or until 10KDa marker is at the bottom of the gel Transfer 1. Make up Tris-Glycine buffer by diluting 5x stock in milliQ H2O and completing to 1X with MeOH so that the final concentration of MeOH is 20%. 2. Cut edges of gel so that all lanes are included 3. Cut two nitrocellulose membranes and incubate in Tris-Glycine buffer until ready to use. 4. Cut whatman gel blot paper slightly smaller than foam pads from the transfer apparatus 5. Prepare sandwich as follows: a. +, foam pad, Whatman Gel Blot Paper, PVDF membrane, Gel, Whatman Gel Blot Paper, foam pad, 6. Load into apparatus. In the space provided, load an ice pack. Run gel at 180mA for 75 minutes

Blotting Buffers: TBS: Tris-buffered saline 960mL milliQ H2O, 10mL 1M Tris, 30mL 5M NaCl TBS/T: 999mL TBS + 1mL Tween-20 5% Milk - TBS/T: 5% powdered milk in TBS/T Protocol: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Rinse membrane TBS. Incubate 2x in TBS for 7 minutes with shaking. Block 45 minutes in 5% Milk TBS/T with shaking Incubate 2x 7 minutes in TBS/T, then 1x 7 minutes in TBS Block 45 minutes in 5% Milk TBS/T with appropriate dilution of primary protein antibody (e.g. 1:10,000 for mouse--tau and mouse--actin) Incubate 2x 7 minutes in TBS/T, then 1x 7 minutes in TBS Block 45 minutes in 5% Milk TBS/T with appropriate dilution of appropriate secondary antibody (e.g. 1:8000 -mouse) Wash 4x 7 minutes in TBS/T Place membrane between the two sheets of plastic of a clear page protector. Make up ECL mix and pipette 1mL over each membrane. Tape page protector with membrane into a film cassette. In a darkroom, load film into cassette. Expose for one minute and load into developer. Expose film to membrane longer as indicated by image on 1 minute exposure.

Transformation of DH5 Cells 1. 2. 3. 4. 5. 6. 7. 8. Make up appropriate number of LB-AMP agar plates. Allow to cool. Add 0.2L of plasmid into 25L of DH5 cells Incubate on ice for 20 minutes Incubate at 42C for 1 minute Incubate on ice for 2 minutes Add 200L of SOC media. Incubate at 32C with shaking for 45 minutes. Move solidified LB-AMP agar plates to 32C incubator to warm before plating Add full volume of transformed bacteria to the solidified surface of an LB-AMP agar plate. Spread evenly across plate with a sterile metal loop. Incubate at 32C for 12 hours. Bacterial Culture for Plasmid Preparation (midiprep, maxiprep) 1. 2. 3. 4. In a falcon tube, add 3mL of LB media and 1.5L of ampicillin Touch a p10 micropipette tip to a single colony and drop into media Incubate tube at 32C with shaking for 8 hours Make up 80mL (midiprep) or 280mL (maxiprep) of LB media. Add 40L (midiprep) or 140L (maxiprep) of ampicillin. 5. Transfer 3mL culture to 80 or 280mL LB-AMP solution. Incubate overnight at 32C with shaking 6. Follow midiprep or maxiprep kit protocol to isolate plasmid from DH5. Typically, the Macherey-Nagel kits were used.