MDA-MB-231 and MCF7 cells were labelled with either Red CMTPX dye or Green CMFDA dye (Invitrogen) according to the manufacturer’s protocol. The color assigned to each of the cell types was randomized for each experiment. Briefly, the media was aspirated from 60–80% confluent cells in a T25. The cells were washed with PBS, and then incubated with 2mL of DMEM containing 1μM CellTracker dye at 37°C for 30min. After incubation, cells were passaged and used for the experiment.
Red cmtpx dye
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
The Red CMTPX Dye is a fluorescent cell tracker dye that can be used to label living cells. It is a cell-permeant dye that upon entering the cell, becomes fluorescent and can be used to monitor cell movement, division, and localization. The dye has an excitation maximum at 577 nm and an emission maximum at 602 nm, which allows for detection using standard fluorescence microscopy or flow cytometry equipment.
Automatically generated - may contain errors
Lab products found in correlation
Green cmfda dye, by Thermo Fisher Scientific (2 mentions)
Lsm 510 microscope, by Zeiss (1 mentions)
Celltracker green cmfda dye, by Thermo Fisher Scientific (1 mentions)
Fv1000 confocal laser scanning microscope, by Olympus (1 mentions)
Ab134131, by Abcam (1 mentions)
Ab17199, by Abcam (1 mentions)
Cell observer spinning disk microscope, by Zeiss (1 mentions)
Tissue tek oct, by Sakura Finetek (1 mentions)
6 protocols using red cmtpx dye
1
Cell Tracking with Fluorescent Dyes
2
Cell Tracking with Fluorescent Dyes
3
Bovine Chondrocyte Visualization and Analysis
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Confocal microscope images of bovine chondrocytes were employed to generate the geometries and finite element meshes. We used 27 original 16-bit grayscale images of the healthy control samples, obtained from the previous research conducted by Lv et al. (2019) (link). These images have a resolution of 2048 × 2048 pixels per slice with a stack size varied between samples. The voxel size is 0.1099 × 0.1099 × 1 µm. The bovine cartilage explants were dyed with a red fluorescent cell tracker (Red CMTPX Dye, Thermo Fisher), cultured in regular medium for four days prior to imaging, and imaged with a Zeiss LSM510 microscope. The benchmark for cell volume estimation in this study was based on the manually estimated cell volume from the previous study, with a detailed procedure provided in the Supplementary Materials .
4
Evaluating IL-1β's Impact on Chondrocyte Volume
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To evaluate the effect of IL-1β on chondrocyte volume, cartilage explants cultured in the regular medium and IL-1β-Supplemented Medium (n = 4 explants from 2 animals per group) were dyed with red fluorescent cell tracker (Red CMTPX Dye, Thermo Fisher) and imaged on a confocal microscope (Zeiss 510) after 4-day culture. The fluorescent image stacks were reconstructed into a 3D image in Image J49 (link). The volume of in situ chondrocytes was registered and quantified (n ≈ 30 cells from each explant). To estimate the cell proliferation rate, primary chondrocytes were extracted from cartilage explants (n = 4 explants from 2 animals per group). MTT assay was then performed following the previous instructions50 (link).
5
Cell Adhesion and Motility Dynamics
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The experimental procedure was performed based on a previously described protocol with modification 14 . Briefly, cells were seeded on 35 mm dishes coated with fibronectin. Upon cell adhesion, cells were stained using CellTrackerTM Green CMFDA Dye or Red CMTPX Dye (Thermo Fisher Scientific) for 30 min. Then, time-lapse images were captured at a total magnification of 200x in 10 min intervals for 500 min using an FV1000 confocal laser scanning microscope (Olympus, Tokyo, Japan). Time-lapse images were analyzed using IMARIS8 software (BitPlane, South Windsor, CT, USA).
6
Multimodal Imaging of Blood Clot Structure
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Cryostat sections from macroscopic blood clots, obtained by tubing loop and embedded in Tissue-Tek O.C.T (Sakura Finetek, Torrance, CA, USA), were fixed in 4% para-formaldehyde and 50% ethanol before analysis. Slides were stained for platelets with an anti-CD41 antibody (rabbit anti-human 1:250, ab134131, Abcam, Cambridge, United Kingdom), for thrombin (mouse anti-human 1:200, ab17199, Abcam), for cell nuclei with 4′,6-diamidino-2-phenylindole (DAPI) and for cells with cell tracker red (Red CMTPX Dye, C34552, Thermo-Fischer Scientific). Four channel images were sequentially collected by a Cell observer Spinning Disk microscope (Zeiss, Oberkochen, Germany), equipped with a Plan-Neofluar 40×/1.30 oil objective and excitation laser lines of 405, 488, 561 and 635 nm, and emission channels at 460/80, 520/35, 617/73 and 685/40 nm.
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