sea water (ASW) (400 mM NaCl, 10 mM KCl, 15 mM HEPES, pH 7.8, 10 mM CaCl, 55 mM MgCl2, and Phenol Red) supplemented to 10 mM N-tert-butyl-alpha-phenylnitrone (PBN) and ,10 mg/mL dispase. Connective tissue was manually re- moved and the intact bag cell cluster was triturated gently with a bent 20 mL pipette tip to separate the neurons, which were arranged on a poly-D-lysine coated, acid washed coverslip. Cultures were maintained for up to 24 hours in L-15 ASW at 18uC prior to use. Fluorescence imaging was performed in L-15 ASW (without Phenol Red) supplemented to 0.25 mM Vitamin E, 3 mg/mL L-carnosine, and 10 mM PBN. Unless noted, cells were maintained in a 1:10,000 dilution of fetal bovine serum from plating onward. Microinjection Microinjection was performed as described previously [21]. For phalloidin injections, 75 mL of methanol phalloidin stock were dried down and resuspended in 4 mL of water for a concentration of 124 mM in the needle. Either Alexa-488 or Alexa-594 con- jugated phalloidin (Molecular Probes) was used in any given experiment. For actin monomer injection experiments, 0.5 mg/ mL aliquots in G-buffer (5 mM Tris, pH 8.1, 0.2 mM CaCl2, 0.2 mM dithiothreitol, and 0.2 mM ATP) were thawed on ice and spun at 20,000 k at 4uC for 30 minutes prior to use. Alexa-488 and/or Alexa-568 rabbit skeletal muscle actin (Molecular Probes) was used in experiments. Lysine-substituted, Texas Red 10 kD dextran (Molecular Probes) was used for volume correction of DNAseI stains. After injection, cells were allowed to recover for at least one hour. Immunocytochemistry Cells were incubated in a flow chamber in Fix (4% formalin, 400 mM NaCl, 10 mM KCl, 15 mM HEPES, pH 7.8, 10 mM CaCl, 55 mM MgCl2, and 400 mM sucrose) for 20–30 minutes and 1% Triton X-100 in Fix for 30 minutes prior to three washes with PBS-T (0.1% Triton X-100, 137 mM NaCl, 2.7 mM KCl, 10 mM phosphate, pH 7.5). For antibody labeling, cells were blocked for 30 minutes in 5% BSA/PBS-T, incubated with primary antibody for 20–30 minutes in 5% BSA/PBS-T, washed three times in 5% BSA/PBS-T, and incubated for 30 minutes to one hour in secondary antibody diluted in 5% BSA/PBS-T. To label actin monomers, 5 mg/mL Oregon Green DNAseI (Molec- ular Probes) in 5% BSA/PBS-T was used after blocking. Cells were washed three times in PBS-T and mounted in MOWIOL media (10% MOWIOL, 25% glycerol, 0.2 M Tris pH 8.5, 20 mM n-propyl-galate). Live Cell Extraction Cells were incubated in a flow chamber for three to five minutes in LE (100 mM PIPES, pH 6.9, 10 mM KCl, 100 mM NaCl, 5 mM EGTA, 5 mM MgCl2, 4% PEG, average MW = 35,000, and ,20% sucrose, ,1000 mOsm/L) supplemented to 1% Triton X-100, ,10 mM phalloidin, 10 mM Taxol, and ,10 mg/ mL BAPTA. TRITC labeled phalloidin (Sigma) was used to visualize actin filaments. For electron microscopy, LE solution was supplemented to 0.1% glutaraldehyde. Extracted cells were washed once with WLE (80 mM PIPES, pH 6.9, 5 mM EDTA, 5 mM MgCl2) and fixed for 20 minutes in 4% formalin/WLE for fluorescence or 5% glutaraldehyde/WLE for electron microscopy. For barbed end labeling experiments extracted cells were incubated in 300 nM Alexa-488-G-Actin (see above) in WLE prior to fixation. If live cell extraction was to be followed by immunocytochemistry, antibody labeling and slide mounting were performed as above. Retrograde Flow Requires Actin Depolymerization Platinum/Palladium Replica Electron Microscopy Cells were prepared essentially as described by Svitkina and Borisy [56], with some modifications. Cells were live cell ex- tracted and fixed as previously described. Often the fixed cells were stored in a humidity chamber overnight at 4uC. A second fixation in 5% glutaraldehyde in water for 20 minutes was followed by 20 minutes in 0.2% tannic acid, three water washes, 20 minutes in 0.2% uranyl acetate, and three water washes. Graded dehydration was performed in increasing concentrations of ethanol (10-20-40-60-80-100-100-100*-100*) prior to critical point drying. The 100* indicates that the ethanol was dried with molecular sieves prior to use. Platinum/palladium rotary shadowing was performed at a 45u angle to a thickness of 2 to 2.5 nm followed by carbon shadowing at a 75u angle. Replicas were mounted on carbon/formvar coated EM grids and observed by transmission EM at 80 kV. Microscopy Images were acquired using an Andor Revolution XD spinning disk confocal built on a Nikon TE 2000E inverted microscope with Perfect Focus System, equipped with an Andor iXonEM+ 888 camera, and CSU-X1 confocal head (Yokogawa). Trans-illumi- nation was provided by a halogen lamp and controlled by a SmartShutter (Sutter Instrument). Confocal excitation was pro- vided by the Andor Laser Combiner System with three laser lines: 488, 561, and 647 nm. Emission wavelength was controlled by a Sutter LB10W-2800 filter wheel outfitted with bandpass filters from Chroma. Image acquisition and all other peripherals were controlled by iQ software (Andor). A 1006, 1.4NA lens was used during all imaging. Quantitative Actin Dynamics Kymography and automated speckle tracking were used to determine rates of movement from time-lapse microscopy. Auto- mated flow tracking was performed with the fsmCenter suite of custom Matlab (MathWorks) algorithms, produced by the Danuser lab, according to the strategy outlined Ji and Danuser [20]. Briefly, an adaptive multi-frame correlation algorithm [20] was run on every 5 or 10 frames to determine the average flow over 5 to 10 frames. This information was used to initialize a single particle tracking algorithm [37,57,58,59], which was run on every frame in the movie. ROI Based Turnover Analysis A custom Matlab (MathWorks) algorithm was written to measure the net kinetic activity within a user defined region of the growth cone. The algorithm is based on conservation of mass principles. The major steps in the analysis are enumerated here and visually in Fig. S1: 1) 2) 3) User selects a region outside of the cell that will be used to determine the average background intensity. The average background is determined separately for each image in the series and is subtracted from any intensity measurements made, in that image, on a pixel by pixel basis. The user defines a region of interest (ROI) in the cell that will be tracked throughout the image series. The spatially average flow, determined within a box around each vertex of the ROI, is calculated based upon the single particle tracking results. The size of the averaging box is user selectable. In this analysis, box sizes were between 33 and 75 pixels wide, depending upon the speckle/vector density of the dataset. Flow averaging is required for two reasons: PLoS ONE | www.plosone.org 12 February 2012 | Volume 7 | Issue 2 | e30959