The linking and bridging capabilities of multi-specific 3WJ/PSMAapt/CD28apt NPs were tested via FACS analysis using CellTracker Green CMFDA and Red CMTPX (Thermo Fisher Scientific). 3WJ/PSMAapt NPs served as a control. Briefly, purified CD8+ T cells were labeled with CellTracker Green CMFDA (1:1,000 dilution), and PSMA-positive LNCap cells (or PSMA-negative PC3 cells) were labeled with CellTracker Red CMTPX (1:1,000 dilution). The CellTracker-labeled cells were incubated at 37°C for 30 min and then washed twice with PBS. Afterward, 105 CD8+ T cells and tumor cells were incubated at a 1:1 ratio at 37°C in Opti-MEM containing 100 nM multi-specific 3WJ/PSMAapt/CD28apt NPs for 2 h. The mixture system was then subjected to flow cytometry analysis using a FACSCalibur flow cytometer. The linking and bridging capabilities of 3WJ/PSMAapt/CD28apt NPs were illustrated by comparing the percentage of double CellTracker-positive cell populations among the different groups. Data were analyzed using FlowJo.
Red cmtpx
Manufactured by Thermo Fisher Scientific
Sourced in United States
Red CMTPX is a fluorescent dye used for cell tracking applications. It is a cell-permeant dye that undergoes a spectral shift upon reaction with thiols, resulting in a red fluorescent signal. The core function of Red CMTPX is to enable the visualization and tracking of live cells.
Automatically generated - may contain errors
Lab products found in correlation
Green cmfda, by Thermo Fisher Scientific (9 mentions)
Celltracker green cmfda, by Thermo Fisher Scientific (8 mentions)
Blue cmac, by Thermo Fisher Scientific (2 mentions)
Ultra low attachment multiwell plates, by Corning (2 mentions)
6 well plastic plates, by Techno Plastic Products (2 mentions)
Eclipse ts100, by Nikon (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Fv1200, by Olympus (1 mentions)
Eclipse te2000 e microscope, by Nikon (1 mentions)
C7025, by Thermo Fisher Scientific (1 mentions)
C34552, by Thermo Fisher Scientific (1 mentions)
Sylgard 184 silicone elastomer kit, by Thermo Fisher Scientific (1 mentions)
C56bl 6 mice, by Jackson ImmunoResearch (1 mentions)
Fv10i liv confocal microscope, by Olympus (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Fluoview software, by Olympus (1 mentions)
Rtci precoated, by Corning (1 mentions)
Notch1 sirna, by Santa Cruz Biotechnology (1 mentions)
Non targeting control sirna, by Santa Cruz Biotechnology (1 mentions)
19 protocols using red cmtpx
1
Multifunctional Nanoparticles for T-Cell Targeting
2
Fluorescent Cell Labeling and Spheroid Staining
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CellTracker™ dyes were purchased from Thermofisher (Green CMFDA, C7025; Red CMTPX, C34552; Blue CMAC, C2110). Cells were seeded 48 h before staining into new flasks. The CellTracker™ dyes were diluted in serum-free medium to final concentrations (Table S3 ) and applied directly to the cells. Images were analyzed using ImageJ and Imaris version 9.6 software (Bitplane, Zurich, Switzerland).
Spheroids have been stained with ethidium homodimer (Thermofisher, Invitrogen, E1169) and calcein (Thermofisher, Invitrogen, C1430) through direct transfer from the culture plate into wells containing the staining solution at the concentration of 10 μM (ethidium homodimer) and 4 µM (calcein). Spheroids were left for ≥30 min in the staining solution till further use.
Spheroids have been stained with ethidium homodimer (Thermofisher, Invitrogen, E1169) and calcein (Thermofisher, Invitrogen, C1430) through direct transfer from the culture plate into wells containing the staining solution at the concentration of 10 μM (ethidium homodimer) and 4 µM (calcein). Spheroids were left for ≥30 min in the staining solution till further use.
3
Inhibition of Angiogenesis by Notch Pathway Modulation
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Briefly, growth factor-depleted Matrigel was thawed overnight at 40 °C and mixed to homogeneity. Culture plates (48-well) were coated with 0.1 ml of Matrigel and allowed to gelatinize at 37 °C for 30 min. HUVECs or HUVECS and HTR8 cells (2.5 × 104 each), labeled with cell tracker Red CMTPX and Green CMFDA (Molecular Probes), respectively, were cultured on Matrigel in the presence of 10% normal pregnancy serum (NPS) containing A-1254 (10 μg/ml). The concentration of A-1254 was selected from a dose response studies described earlier11 (link). The endothelial cell tube formation was monitored and recorded after 12–14 hrs of incubation using the florescence microscopy at 4 x magnification (Nikon Eclipse TS 100 coupled with CCD camera) as described11 (link)33 (link). To evaluate the significance of the Notch/Dll pathway in A-1254-induced inhibition of angiogenesis, A-1254-induced disruption of three-dimensional tube formation by HUVECs was assessed in the presence or absence of different doses of γ-secretase inhibitor, L1790 (0, 1, 5, 10, 25 μM) (Sigma-Aldrich).
4
Confocal Microscopy for Live/Dead Cell Imaging
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Example 4
Methodology for Confocal Microscopy
Leica TCS SP5II HCS A confocal images taken with HC PL APO 10×/0.4 objective using Red (HeNe 633 nm/12 mW) and Lime (DPSS 561 nm/20 mW) lasers with PTM detectors. Live/Dead imaging was performed with fluorescein diacetate (FDA) and propidium iodide (PI) cellular stains (Molecular Probes®, USA) for live and dead cells respectively. Cell co-culture imaging was carried out with dyes CellTracker™ Green CMFDA and Red CMTPX (Molecular Probes®, USA) for HepG2 and SK-HEP-1 cells respectively.
5
Confocal Microscopy Analysis of NFCA Threads
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NFCA threads and related cell studies were examined and analyzed with confocal microscopy (Leica TCS SP5II HCS A, Leica Microsystems, Germany). Images were taken with HC PL APO 10x/0.4 objective using Red (HeNe 633 nm/12mW) and Lime (DPSS 561 nm/20mW) lasers with PTM detectors. Live/Dead imaging was performed with fluorescein diacetate (FDA) and propidium iodide (PI) cellular stains (Molecular Probes®, USA) for live and dead cells, respectively. Cell co-culture imaging was carried out with cellular dyes CellTracker™ Green CMFDA and Red CMTPX (Molecular Probes®, USA) for SK-HEP-1 and HepG2 cells, respectively. Suture and NFCA coating diameters (5 x 20 cm sutures) were measured using Leica LAS AF lite imaging suite (Leica Microsystems, Germany). 2 random positions were selected from each coated suture and diameter measured (n = 10).
6
Cell Staining for 3D Co-culture Imaging
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Prior to cell harvesting, CRC cells, fibroblasts and endothelial cells were washed with PBS and incubated in serum free medium for 20 minutes supplemented with 40 µM blue CMAC, 5 µM Green CMFDA or 10 µM Red CMTPX (C2110, C7025 and C34552, Life Technologies), respectively. Afterwards, 3D co-cultures were established and fluorescent imaging was performed.
7
Fluorescence Microscopy of Labeled Cells
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For fluorescence observation, cells were fixed with 1% paraformaldehyde (Wako, Japan) for 30 min, washed with PBS (Gibco Invitrogen, Grand Island, NY, USA), incubated with the 10-µM cell tracker Red CMTPX (Life Technologies, USA) in DMSO for 15 min at 25°C. Thereafter, cells were washed with PBS, and then incubated with 0.7 µg/ml Hoechst 33342 (Invitrogen, USA) for 15 min. Fluorescence images and phase contrast images were acquired using a highly sensitive color camera (DP-73, Olympus, Japan) attached to an inverted fluorescent microscope (IX-71, Olympus, Japan). Cross sectional images of cells were obtained using a confocal microscope (FV-1200, Olympus, Japan) with a set of lasers (405, 473, and 543 nm).
8
Chemotactic Assay of Fibroblast Migration
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Chemotactic assays were performed as described previously (Suraneni et al., 2012 (link)), with several modifications. Briefly, ARPC3+/+ or ARPC3−/− fibroblast cells were trypsinized, diluted to ∼2 × 106 cells/ml, and plated in a μ-Slide Chemotaxis slides (Ibidi, Martinsried, Germany) precoated with 5 μg/ml of fibronectin and allowed to recover for 5–6 h. The medium was replaced with low-serum medium (DMEM with 0.5% FBS) overnight, followed by replacement with fibroblast medium. Then one of the ports was filled with chemoattractant (500 ng/ml PDGF or EGF) solution. Cell migration in response to chemotactic signal was recorded by placing the μ-Slide on an inverted Nikon Eclipse TE2000-E microscope with a 37°C incubator and 5% CO2 for a period of 12 h with frames taken every 20 min. Cell-trajectory analysis was performed as described previously (Suraneni et al., 2012 (link)).
For the mixed-cells experiment, wt and mutant fibroblasts were labeled with live-cell tracker green CMFDA (C7025; Life Technologies, Grand Island, NY) and red CMTPX (C34552; Life Technologies), respectively, before trypsinization and mixing. The ARPC3+/+ cells with ARPC3−/− cells conditional medium experiments were performed by collecting the medium from ARPC3−/− cell dishes after 12 h of culturing. The remaining procedure was performed as described in the preceding section.
For the mixed-cells experiment, wt and mutant fibroblasts were labeled with live-cell tracker green CMFDA (C7025; Life Technologies, Grand Island, NY) and red CMTPX (C34552; Life Technologies), respectively, before trypsinization and mixing. The ARPC3+/+ cells with ARPC3−/− cells conditional medium experiments were performed by collecting the medium from ARPC3−/− cell dishes after 12 h of culturing. The remaining procedure was performed as described in the preceding section.
9
Murine Peritoneal Adhesion Assay
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Cell-to-peritoneum adhesion was assessed ex vivo as published previously [40] (link), [41] (link). Briefly, C56BL6 mice (Jackson Laboratories) were euthanized by CO2 inhalation and subsequent cervical dislocation, then dissected using a ventral midline incision; four peritoneal tissue pieces were removed and pinned to the bottom of 24-well dishes precoated with optically transparent silicone using Sylgard 184 Silicone Elastomer Kit (Fisher). OvCa433Ncad+ and SKOV3Ecad+ cells were transiently stained with red CMTPX or green CMFDA (Invitrogen), respectively. Parental OvCa433 and SKOV3 cells were RFP- or GFP-labeled, respectively. Cells were applied to murine peritoneal explants, incubated for 1 to 2 hours, and washed with 3 × 3-minute ice-cold PBS washes, and cells/peritoneum were imaged with AMG EVOS fluorescence microscope. Image analysis was performed with ImageJ. The assay was repeated in triplicate, and statistical analysis was performed using a Student's t test. The fourth replicate tissue explants were subjected to SEM processing and imaging as described above.
10
Co-Culturing Cells and Microcapsules in PDMS Microwell
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To facilitate the observation of cellular distribution, Celltracker™ Green CMFDA and Red CMTPX (Invitrogen, c34552) were used to stain A549 cells and HUVECs, respectively, following the manufacturer's instructions. Before seeding, A549, HUVECs, and 3T3 cells were digested and resuspended to a cell density of 5 × 105 cells/mL. The concentration of microcapsules was 5000/mL, and A549, HUVECs, and 3T3 cells were co-cultured in the ratio of 9:9:2 in a PDMS-based microwell device following previously established protocols [38 ,39 (link)]. Honeycomb microwell arrays of diameter 326 µm were prepared by casting PDMS on silicon wafers. The resulting PDMS membrane was peeled off, treated with hydrophobic agents, cut, and fixed in a 24-well culture plate for cell inoculation. The cells and microcapsules were cultured for 2 days before conducting the follow-up experiments.
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