LR73 cells were transiently transfected with the indicated plasmids on the 18-mm Ø cover glasses in a 12-well plate. One day after transfection, the cells were incubated with either 1 μl of 2 μm carboxylate-modified red fluorescent beads (Invitrogen) or TAMRA-stained apoptotic thymocytes in a CO2 incubator at 37 oC for 2 h. The cells were then fixed with 4% paraformaldehyde in PBS for 15 min at room temperature (RT) and rinsed with PBS twice for 3 min. Then, the cells were blocked with blocking solution containing 3% bovine serum albumin (BSA, Bovogen, Keilor East, VIC, Australia), 0.1% Triton X-100 (Usb), 0.1% sodium azide (Sigma) and 1% HINGS (Gibco, Waltham, MA, USA) in PBS for 30 min at RT. Next, the cells were incubated with primary antibody (anti-HA, Santa Cruz, F-7) in 3% BSA in PBS at 4 oC overnight. The cells then were washed with PBS for 5 min twice and incubated with secondary antibody (goat anti-mouse Alexa fluor 488, Life Technologies) for 1 h at RT. The cells were rinsed with PBS for 5 min twice and incubated with Hoechst 33342 (Invitrogen). Images were acquired using either Zeiss Axio Imager D2 or Zeiss LSM 700 (Oberkochen, Germany).
Carboxylate modified red fluorescent beads
Manufactured by Thermo Fisher Scientific
Carboxylate-modified red fluorescent beads are spherical particles composed of a polymer matrix that contains a red fluorescent dye. These beads are designed to emit a red fluorescent signal when excited by a light source of appropriate wavelength.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 700, by Zeiss (2 mentions)
Matlab, by MathWorks (2 mentions)
Bovine serum albumin (bsa), by Santa Cruz Biotechnology (1 mentions)
Axio imager d2, by Zeiss (1 mentions)
Bovine serum albumin (bsa), by Bovogen (1 mentions)
Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Anti ha, by Santa Cruz Biotechnology (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Sodium azide, by Merck Group (1 mentions)
Canto 2, by BD (1 mentions)
Acrylamide bis solution, by Bio-Rad (1 mentions)
Multiphysics, by Comsol (1 mentions)
Temed, by Bio-Rad (1 mentions)
Purecol, by Advanced BioMatrix (1 mentions)
Glass coverslip, by Matsunami (1 mentions)
6 protocols using carboxylate modified red fluorescent beads
1
Phagocytosis Assay in Transfected Cells
2
Phagocytosis Assay for Cell Lines
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The 1 × 105 LR73 cells were plated on a 24-well culture plate and transfected with GFP alone or GFP and the indicated plasmids using Lipofectamine 2000. One day after transfection, the cells were incubated with 1 μl of carboxylate-modified red fluorescent beads (2 μm in diameter) (Invitrogen) or 2 × 106 TAMRA stained apoptotic thymocytes for 2 h, trypsinized, and analyzed by flow cytometry (Canto II, BD). GFP and red-fluorescent double-positive cells were classified as phagocytes engulfing carboxylate beads or apoptotic thymocytes. To make TAMRA-stained apoptotic thymocytes, thymocytes were prepared from thymi of 5 to 8 week-old C57/B6 wild type mice, and stained with 50 μM TAMRA. To induce apoptosis of thymocytes, cells cultured in RPMI 1640 medium (containing 10% FBS and 1% penicillin-streptomycin-glutamine) were stimulated with 50 μM dexamethasone (Calbiochem) in a 5% CO2 incubator for 4 h, washed with phagocyte culture medium twice, and resuspended with phagocyte culture medium at a concentration of 2 × 106 cells/300 μl.
3
Phagocytosis Assay for Cultured Cells
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LR73 cells or L cells on 24-well plates were transfected with the indicated plasmids. One day after transfection, engulfment assays were performed as follows. Transfected cells were incubated with either 2 μm carboxylate-modified red fluorescent beads (Invitrogen) or TAMRA-labeled apoptotic thymocytes in an incubator with 5% CO2 at 37 °C for 2 h. Next, the phagocytes were extensively washed with ice-cold PBS, trypsinized, resuspended, and analyzed by flow cytometry. Transfected cells were identified by GFP fluorescence, and engulfment targets were identified by red fluorescence. Double-positive cells were considered to represent phagocytes engulfing targets. Flow cytometry data were analyzed using the FlowJo software.
4
Traction Force Microscopy for Contractile Forces
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Traction force microscopy was used to quantify the contractile forces generated by MMFs as described in [36] (link). Briefly, polyacrylamide (PA) gels with a Young's modulus of 5.9 kPa and embedded with 0.5 μm diameter red fluorescent carboxylate-modified beads (Invitrogen, Carlsbad, CA) were prepared on 25 mm glass coverslips. These gels were coated with bovine collagen type I using heterobifunctional crosslinker sulfo-SANPAH. MMFs were sparsely seeded on the gels and cultured for 24 hours before imaging. Inverted fluorescent microscope (Olympus IX81, Olympus Corporation, Tokyo, Japan) was used to obtain phase contrast images of the single cell boundaries as well as the fluorescent images of the underlying beads before and after the cell detachment using trypsin. The bead displacement between the images with and without cells was computed using correlation-based particle image velocimetry (MATLAB, MathWorks, Natick, MA). These displacement data along with the cell boundaries were then used to compute the individual cell tractions using finite element model in Comsol (COMSOL Multiphysics, Burlington, MA) as described previously [37] (link).
5
Contractility Measurement of Fibroblasts
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Traction force microscopy was used to measure the contractility of the fibroblasts at individual cell level as described previously [26 (link)]. Cells were maintained under normal culture conditions. TFM substrates were prepared with polyacrylamide (PA) on 22 mm glass coverslips using an 8% acrylamide and 0.04% bis-acrylamide solution (BioRad) polymerized with 10% ammonium persulphate and tetramethylethylenediamine (TEMED). The gels were embedded with 0.5 μm-diameter red fluorescent carboxylate modified beads (Invitrogen) and coated with 50 μg/mL bovine collagen type I (Advanced BioMatrix Inc.). Cells were seeded (~1000/gel) in their respective treatment media and allowed to adhere overnight. After 24 hours, isolated single cells were imaged using phase contrast microscopy to identify cell boundaries, while fluorescent images of bead positions were obtained before and after cell detachment by trypsinization. Images were then analyzed to compute the bead displacement along the cell boundary using correlation-based particle image velocimetry in MATLAB (MathWorks). The traction stress field on the gel surface was calculated using the 3D finite element software COMSOL Multiphysics where the measured bead displacements under the cell were applied as a boundary condition, and the resulting stress field was analyzed for average traction and net contractile moment [26 (link)].
6
Polyacrylamide Gel Substrate Preparation
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Polyacrylamide gel substrates were prepared according to the previously published protocols [8 (link), 9 (link)]. Briefly, the gel solution was prepared with 3% acrylamide, 0.25% bisacrylamide, 0.8% ammonium persulfate, 0.08% TEMED (Bio-Rad products), and 0.01% red fluorescent carboxylate-modified beads (0.5μm diameter, Invitrogen). 20 μl of this mixture was added to each dish and the samples were covered with glass cover slips with 18 mm diameter (Matsunami). After the polymerization, the surface was coated with type I collagen (Purecol, Advanced BioMatrix) using 4 μM sulphosuccinimidyl-6-(4-azido-2-nitrophenylamino) hexanoate (Sulfo-SANPAH; Pierce). Young’s modulus of the gel was characterized by the conventional method using the Hertz equation [16 ], obtaining E = 2500±600 Pa. The Fourier-transform traction microscopy [8 (link)] was used to estimate traction force fields from bead displacement fields.
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