MSCs were stained with red fluorescence using CellTracker CMTPX (Thermo, Waltham, MA, USA). Briefly, harvested and resuspended cells were gently mixed with pre-warmed CellTracker CMTPX (at 1 μM/2 × 106 cells). Then, cells were incubated for 20 min at 37 °C.
Celltracker cmtpx
Manufactured by Thermo Fisher Scientific
Sourced in United States
CellTracker CMTPX is a cell-permeant dye that is modified to contain a chloromethyl group. It can be used to label cells for long-term tracking and lineage analysis.
Automatically generated - may contain errors
Lab products found in correlation
5 protocols using celltracker cmtpx
1
Fluorescent Labeling of MSCs
2
hASC Biodistribution in Ischemic Injury
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hASCs (2 × 106 hASCs in 0.5 ml of EBM‐2/1%BSA) were labelled using Celltracker CMTPX (Thermofisher.com) according to manufacture instructions and were injected into the suprarenal aorta at the time of reperfusion. Kidney, spleen, lung and hindlimb skeletal muscles were harvested at 10 min. and 48 hrs post‐injection, then immediately sectioned without fixation and evaluated for the presence of fluorescent cells using confocal microscopy (Olympus FV 1000‐MPE microscope, Center Valley, PA, USA).
3
Visualizing Immune Cell Interactions
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ilnFRCs encapsulated into PEG gels and cultured for 14 days were stained with 25 µM CellTracker CMFDA (Invitrogen) by direct addition of the dye to the gels. Following incubation for 45 min at 37 °C, the gels containing encapsulated ilnFRCs were washed with PBS twice. In parallel, A3.01T cells were stained by 12.5 µM CellTracker CMTPX (Invitrogen) for 30 min at 37 °C and washed with PBS twice. The stained A3.01T cells were added to the PEG gels encapsulating stained ilnFRCs and cocultured for 3 days. The cells were visualized using a Nikon A1 confocal microscope with a ×10 objective lens (Nikon). Z-series of images were acquired with 5.92 µm intervals between focal planes. A maximum intensity projection image and a 3D reconstructed image of the z-series images composed of 40 focal planes were obtained with ImageJ software (NIH; downloaded from http://rsbweb.nih.gov/ij/ ).
4
Cell Labeling with CellTracker CMTPX
5
Spheroid Formation and Multicolor Imaging
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To generate spheroids for the invasion experiments, 105 isolated cells were plated in a 6-well plate with gentle rotational shaking at approximately 34 rpm and allowed to form spheroids for 2 d in the presence of either CellTracker CMTPX (Red dye, Invitrogen, Carlsbad, CA, USA), CMAC (Blue dye, Invitrogen, Carlsbad, CA, USA) or CMFDA (Green dye, Invitrogen, Carlsbad, CA, USA), respectively (Figure 1 a). Combination was achieved by transferring labeled spheroids into a 2.0 mL Eppendorf tube using a sterile plastic eyedropper, washing once in PBS, then combining the spheroids of interest at the bottom of a 1.5 mL Eppendorf tube. The spheroids were allowed to settle at the bottom of an Eppendorf tube followed by a 1 h incubation at 37 °C to allow contact adhesion to occur. For live cell imaging, aggregates were mounted in 0.8% Noble agar/PBS that had been cooled to 39 °C prior to imaging in a 35 mm glass- bottom dish (MatTek Corporation, Ashland, MA, USA). A 37 °C heater was used to warm the imaging chamber, beginning at least 3 h prior to the start of live cell imaging. Aggregated spheroids were imaged at single cell resolution with a Leica TCS SP5 II Basic VIS confocal system (Leica Microsystems, Wetzlar, Germany) at a time interval of 10 min for 2 h.
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