Cells were treated with control, TNF-α alone (50 ng/ml) or TNF-α with inhibitor (1 mM PDTC, 10 µM U0126 or 20 mM SP600125). MSC adherence to HUVECs was measured using fluorescent carbocyanine CM-Dil (Invitrogen; Thermo Fisher Scientific, Inc.) to label the cell membranes. Minor modifications were made to the manufacturer's protocol. Briefly, prior to transplantation, MSCs were labeled with 2 µg/ml CM-Dil for ~30 min at 37°C, then washed three times with phosphate-buffered saline (Thermo Fisher Scientific, Inc.) and transferred to a 6-well plate containing HUVECs for 12 h. Non-adherent cells were removed by washing with medium. The number of adherent cells were counted in 5 fields of each sample (n=4) at x4 magnification (BX51M; Olympus Corporation, Tokyo, Japan).
Cm dil
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
CM-Dil is a fluorescent cell membrane dye used for the labeling and tracking of live cells. It binds to the cell membrane and is retained during cell division, allowing for the visualization and monitoring of cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 780 confocal microscope, by Zeiss (6 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Lsm 710, by Zeiss (3 mentions)
Cm dil, by Olympus (3 mentions)
Exo glow exosome labeling kit, by System Biosciences (3 mentions)
Tricaine, by Merck Group (3 mentions)
Fluorescence microscope, by Olympus (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Bx51m, by Olympus (2 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Confocal microscope, by Leica (2 mentions)
Total exosomes isolation kit, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Olympus (2 mentions)
Calcein am, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Im 300, by Narishige (2 mentions)
Exoquick, by System Biosciences (2 mentions)
Sprague dawley rat, by Charles River Laboratories (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
99 protocols using cm dil
1
MSC Adhesion to HUVECs under Inflammatory Conditions
2
Fluorescent Tracking of Xenografted Cells
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For xenograft studies, two approaches of fluorescent cell tracking were utilized and their staining intensities were analyzed in vitro prior to transplantation. The sorted Y79 cells were labeled using lipophilic carbocyanine red dye CM-Dil (ThermoFisher Scientific, USA). Briefly, the cells were washed and resuspended in 1 mL serum free media. The cells were stained with 5 mL of CM-Dil (ThermoFisher Scientific, USA) and incubated for 20 min at 37°C. Post labeling, the cells were repeatedly washed with serum free media to remove any unconjugated dye. The labeled cells were then incubated for 2 h and assessed for staining efficiency using a fluorescence microscope prior to transplantation. The cells were also evaluated for dye retention in vitro for over 2 weeks.
3
Tracking Mesenchymal Stem Cell Tropism
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To evaluate the tropism of MSCs for cancer cells in vivo, AF-MSCs and IFNα-AF-MSCs were labeled with a fluorescent cell surface marker, CM-Dil (Invitrogen), by incubation with a working solution of 5 μg/ml CM-Dil for 5 min at room temperature, followed by a 20-minute incubation at 4°C. After labeling, the cells were washed with PBS and cultivated in fresh medium prior to injection into mice, which occurred within 24 h.
4
Isolation and Cryopreservation of Rat Hepatocytes
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Hepatocytes were isolated from 250 to 350 g Sprague Dawley rats (Charles River, Harlow, UK), by in situ collagenase perfusion of the liver as previously described [27 (link)]. The cell suspension was centrifuged at 50×g for 5 min at 4 °C to isolate the hepatocytes. Cell viability was determined using trypan blue and always reached at least 65%. Cells were cryopreserved at 1 × 107 cells/ml in UW solution with 5% glucose and 10% DMSO and thawed at time of transplant [28 (link)]. To allow cell tracking, the transplanted cells were stained with 60 μM CM-DiL, a lipophilic carbocyanine cell tracker dye, excitation 553 nm, emission 570 nm (Thermo Fisher Scientific, Paisley, UK). Cells were incubated with the CM-DiL dye at 37 °C for 5 min and then at 4 °C for 15 min. Unbound dye was washed off by centrifugation at 50×g for 5 min. In addition, eGFP rat hepatocytes were isolated from Lewis-Tg (CAG-eGFP) Ysrrc rats and are known as HepaCur™. The cells were kindly donated by Yecuris, Portland, OR, USA.
5
Labeling hUC-MSCs with CM-Dil
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Add culture medium containing 2 mg/L CM-Dil (Thermo Fisher Scientific Inc., Waltham, MA, USA) when hUC-MSCs reach 80% confluency after two rinses with sterile PBS. Then incubate for 10 min at 37 °C and 15 min at 4 °C. Next, the labeled cells were monitored for fluorescence using the Olympus BX51 microscope (Olympus, Tokyo, Japan) followed by two washes with sterile PBS. Subsequently, the cells were detached and subcultured. Viability and proliferation of hUC-MSCs and CM-Dil-labeled hUC-MSCs at P3 were analyzed with the MTT method, according to the manufacturer’s instructions. They were seeded in 96-well plates at a concentration of 5 × 103 cells/well in the FBS medium. The assay was performed every 24 h during 7 days. The absorbance was measured at a wavelength of 570 nm by using a spectrophotometric plate reader (Mithras LB 940, Berthold Technology, BadWildbad, Germany).
6
Zebrafish Xenograft Metastasis Assay
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GC cells were labeled with CM-Dil (Thermo Fisher Scientific, Waltham, MA) using the manufacturer's protocol then washed in phosphate-buffered saline and resuspended at 10,000 cells/mL. Zebrafish embryos at 48 h post-fertilization were anesthetized by placement in 0.04 mg/mL of ethyl 3-aminobenzoate methanesulfonate (tricaine). CM-Dil cells (100 cells in 10 nL; 30 nL injection volume per embryo) were injected with a pneumatic picopump injector using glass microinjection needles. Groups of larvae (30) with red fluorescence at the injection site were moved to 6-well plates (30 embryos per well). Larvae were treated at 8 days post-injection (dpi) and incubated at 35°C during treatment.
Stock solutions of Huaier to be tested were made at a 100× working concentration in an appropriate solvent. Huaier was then diluted to the finalconcentration with E3 medium. Fresh drug-containing E3 medium was replaced daily;E3 medium alone was used on untreated controls. We evaluated tumor growth and metastasis in injected zebrafish larvae every 2 dpi by fluorescence microscopy. Images were captured on a Nikon SMZ1500 microscope, and the distance of metastatic spread was measured using NIS-Elements D 3.10 software. The relative distance of metastasis was calculated using the following formula: relative distance=distance at each point/distance at 0 dpi.
Stock solutions of Huaier to be tested were made at a 100× working concentration in an appropriate solvent. Huaier was then diluted to the finalconcentration with E3 medium. Fresh drug-containing E3 medium was replaced daily;E3 medium alone was used on untreated controls. We evaluated tumor growth and metastasis in injected zebrafish larvae every 2 dpi by fluorescence microscopy. Images were captured on a Nikon SMZ1500 microscope, and the distance of metastatic spread was measured using NIS-Elements D 3.10 software. The relative distance of metastasis was calculated using the following formula: relative distance=distance at each point/distance at 0 dpi.
7
Tracking Transplanted Adipose-Derived Stem Cells
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To track the localization of transplanted ADSCs, approximately 1 × 106 ADSCs were added with 1 ml of culture medium containing 2 mg CM-Dil (Thermo, USA) and kept at 37 °C for 10 min and 4 °C for 20 min. Subsequently, the labeled cells were rinsed twice with sterile PBS to remove the unbound CM-Dil and suspended in PBS for subcutaneous transplantation with ovarian fragments (6 × 106 cells per rat) or thoroughly mixed with “bioinks” (1 × 107 cells ml−1 “bioinks”, 0.6 ml “bioinks” per entire rat) for printing. The bilateral grafts were removed at 1 and 4 weeks after transplantation, fixed with optimal cutting temperature (OCT) compound, cut into 6 µm in thickness using a cryostat (Leica, Germany), incubated with DAPI and subsequently imaged under a fluorescence microscope (Olympus, Japan) to examine the survival and distribution of ADSCs in vivo.
8
Xenograft Inhibition Assay in Zebrafish
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AB strain zebrafish was used to study the inhibition of PELA54-CD/DOX on the growth of xenografts of MCF-7/ADR cells. Approximately 400–500 MCF-7/ADR cells labeled with CM-Dil (V-22885; Thermo Fisher Scientific) were microinjected into the yolk sac of 48 hpf embryos. PELA54-CD/DOX, DOX·HCl, or PBS as blank was microinjected into the pericardium 24 hours later. Anesthetized zebrafish were imaged both before and 48 hours after injection of different drugs. The fluorescent signal (S) obtained from images represented the growth of MCF-7/ADR cells, and the inhibition rate was calculated using the equation: Inhibition rate of growth (%) = (1 − SPELA54-CD/DOX/Scontrol) × 100. Results are presented as mean ± SE from ten independently performed experiments.
9
Tracking Endometrial Stem Cells Transplantation
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After magnetic microbeads selection, the eMSC were incubated in PBS containing 2 mg/mL CM-Dil (Thermo Fisher Inc., Waltham, MA, USA) at 37oC for 5 min, followed by a 15-min incubation at 4oC. Labeling efficiency was examined under a fluorescence microscope and over 90% of the cell population was red-fluorescent cells (Supplementary Fig S1 C). After intrauterine transplantation of CM-Dil labeled eMSC, uterine horns were harvested at post-operative Day 3, 7 and 14 and frozen in optimal cutting temperature (OCT) compound (Sakura Finetek, Torrance, CA, USA) in liquid nitrogen and stored at -80o C until required. Frozen sectioned at 5 μm thickness were stained with dilution factor l in 1000 DAPI (Invitrogen) for 1 min to visualize the location and migration of eMSC in vivo.
10
Subcellular Localization of RrTTG1 Protein
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The CDS of RrTTG1 was cloned in-frame into the XbaI-digested pM999-eGFP plasmid, which contains an enhancer green fluorescent protein (eGFP) tag driven by CaMV 35S promoter. The constructed and empty vector plasmids were independently transformed into suspension protoplasts of Arabidopsis by PEG (polyethylene glycol) transformation (Yan et al., 2021 ). The fusion protein localization was monitored in the next 24 h. The nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI; Roche, Switzerland) at 0.1 μg/mL for 10 min. Meanwhile, the cell membrane was labeled by chloromethyl-benzamidodialkyl carbocyanine (CM-Dil, Thermo Fisher Scientific, United States) at 5 μg/mL for 15 min. The fluorescence was visualized using a laser confocal scanning microscope (SP8, Leica, Germany).
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