Metamorph 7

Non-treated THP-1 cells and primary monocytes were washed and resuspended in PBS and laid on coverslips pretreated with poly-L-lysine (0.1 mg/mL) (Sigma) for 20 min at room temperature. PMA-treated THP-1 and monocyte-derived macrophages (MDMs) were grown on non-treated coverslips, washed in PBS, and then fixed with 4% paraformaldehyde in PBS. Intracellular PLSCR1 was detected with the 1E9 mAb in 1% BSA-PBS, supplemented with 0.1% Triton X-100 for permeabilization, and then stained using Alexa488-coupled secondary antibody. Cell surface PLSCR1 was detected either with the 1E9 mAb or with the rabbit pAb. Samples were examined under an epifluorescence microscope (Leica DMB) with a cooled charge-coupled device camera (Micromax 1300Y/HS; Roper Princeton Instruments), using a Plan APO 100X objective. Images acquisition was perfomed with MetaMorph 7.6 (Molecular Devices). After culturing BMDMs for at least 10 days in 10 ng/ml mCSF (Miltenyi Biotec), cells were plated on non-treated coverslips, subsequently fixed and permeabilized as described above, and then labeled with TRITC-phalloidin (Sigma) and rabbit anti-Iba1 (Wako Chemicals GmbH) and an Alexa 647 coupled-goat anti-rabbit-IgG (Molecular Probes). Images were acquired using a confocal microscope (Leica SP5II) equipped with a Plan APO 63x objective (N.A. 1.40, pinhole 1.0).

DIC images were analyzed morphometrically employing MetaMorph 7.6 software (Molecular Devices, Sunnyvale, CA). E14 and E16 neurons after 4 h treatment with or without netrin-1 were subjected to the analysis of axon branching/outgrowth [11 (link)]: the lengths of primary axon shafts (excluding growth cones) were measured and the numbers of branch points (with branches longer than 12 μm and/or lamellipodium-tipped, [11 (link)]) were counted along the whole length of each primary axon. E14 and E16 neurons after 30 min treatment were employed for the analysis of filopodial protrusion on the axon shafts [11 (link)]: in addition to the measurement of the lengths of primary axon shafts, the numbers of filopodial protrusions (12 μm or shorter and lacking a lamellipodial tip) were counted along each primary axon. Following Jarque–Bera test for normality, nonparametric statistical analyses were performed on the data: Steel–Dwass test or Wilcoxon rank sum test was employed where appropriate. Correlation between axon length and the density of branches was analyzed using Spearman’s rank correlation test, which was applied for the primary axons with at least one branch. Differences were considered significant if p < 0.05.

HEK293 cells were cultured in a 35-mm coverslip bottom dish or a 12-well plate to obtain images and measure FRET efficiency. To obtain the image and FRET efficiency of a cell, we used an inverted microscope with a 60x oil objective lens and the three-cube FRET calculation^{61 (link),62 (link)} controlled by MetaMorph 7.6 (Molecular Devices, U.S.A). We mainly used three-cube FRET and mCherry (FF01-562/40, FF593-Di03, FF01-617/75, Semrock). The three-cube FRET efficiency (cube settings for CFP, YFP, and Raw FRET) was acquired from a pE-1 Main Unit to three-cube FRET (excitation, dichroic mirror, filter) through a fixed collimator: CFP (ET 435/20 nm, ET CFP/YFP/mCherry beam splitter, ET 470/24 nm, Chroma); YFP (ET 500/20 nm, ET CFP/YFP/mCherry beam splitter, ET 535/30 nm, Chroma); and CFP/YFP FRET (ET435/20 nm, ET CFP/YFP/mCherry beam splitter, ET535/30 nm, Chroma). The excitation LED and filter were sequentially rotated, the rotation period for each of the filter cubes was ~0.5 s, and all images (three for CFP/YFP/Raw FRET) were obtained within 2 s. Each of the images was acquired on a cooled 3 MHz (14 bit) EMCCD camera (iXon Ultra 888: ANDOR) with an exposure time of 100 ms with 1 × 1, 2 × 2, or 3 × 3 binning under the control of MetaMorph 7.6 software. Our FRET recording of the fluorophores was restricted in a range of CFP/YFP ratio from 0.5 to 2.0.

Fixed cells were incubated for 10 min with 0.25% Triton X-100 followed by 1% albumin overnight at 4°C for blocking. Primary paxillin antibody (1∶2000, ab32084, Abcam) was applied for 2 hours at room temperature, and then a secondary AlexaFluor 488-conjugated antibody (1∶2000, Invitrogen) was applied for 1 hour or rhodamine phalloidin (1∶2000 Invitrogen) and Hoechst 33342 (3.2 µM, Invitrogen) for 30 min at room temperature. The cells were subsequently mounted with Fluoromount-G (Southern Biotech, Birmingham, AL). All buffers used contained 1 mM MgCl₂. The samples were imaged by using a CARV II confocal (BD Biosciences) Nikon Eclipse Ti-S microscope equipped with a motorized, programmable stage using a Cool-Snap HQ camera (Photometrics) and controlled by Metamorph 7.6 (Molecular Devices). A custom-written MATLAB (Mathworks) program was used to quantify cell area and focal adhesion number and size.