HUVECs were transfected according to the previous method’s description and in vivo stained, according to the company protocol, with CellTracker™ Green CMFDA Dye (Lie Technologies, Carlsbad, CA, USA and then seeded overnight in a 96-wells plate at the density of 5000 cells/well. The experiment was simultaneously performed in either standard culture conditions (21% O2) or hypoxia (1% O2). The following day, pericytes were stained according to the company protocol with CellTracker™ CM-DiI Dye (Life Technologies) and left for 2 h in full media. The HUVEC-containing wells were then gently washed with medium before seeding of 5000 pericytes on the HUVECs layer. Photomicrographs (40X) were taken at 12h from pericyte seeding. For each treatment, five photomicrographs were taken, and each treatment was performed in triplicate. ImageJ was used to quantify the adhesion.
Celltracker cm dii dye
Manufactured by Thermo Fisher Scientific
Sourced in United States
CellTracker CM-DiI Dye is a fluorescent cell labeling reagent used for tracking and visualizing cells. It is a lipophilic carbocyanine dye that passively diffuses into live cells and becomes incorporated into the cell membrane. The dye is retained within the cell during cell division, allowing the monitoring of cell lineage and migration.
Automatically generated - may contain errors
Lab products found in correlation
Dmem f12, by Thermo Fisher Scientific (3 mentions)
Diaphorase, by Merck Group (2 mentions)
Glutamine, by Thermo Fisher Scientific (2 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (2 mentions)
Ap20187, by Takara Bio (2 mentions)
Recombinant mouse fcγr 1 and fcγr 4, by ACROBiosystems (2 mentions)
Mabselect sure resin, by Merck Group (2 mentions)
Superdex 200i 10x300gl column, by Merck Group (2 mentions)
Recombinant human and mouse cd38 proteins, by R&D Systems (2 mentions)
Nmnat1, by R&D Systems (2 mentions)
Anti mouse tnfα and il6 neutralizing antibodies, by R&D Systems (2 mentions)
Luminex premixed multi analyte magnetic luminex assay kit, by R&D Systems (2 mentions)
α mem, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Histopaque 1077, by Merck Group (2 mentions)
Olaparib, by LC Laboratories (2 mentions)
Collagenase type 2, by Worthington (2 mentions)
Quikchange site directed mutagenesis kit, by Agilent Technologies (2 mentions)
39 protocols using celltracker cm dii dye
1
Pericyte Adhesion to HUVECs under Normoxia and Hypoxia
2
Isolation and Characterization of Circulating Tumor Cells
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Streptavidin coated magnetic beads were purchased from Bangs Laboratories (catalogue-CM01N). Dynabeads MyOne Streptavidin T1 beads were purchased from Invitrogen (catalogue-65601). Biotin-labeled anti-human EpCAM antibody was obtained from BioLegend (Catalog #324,215). Cell culture medium RPMI-1640, CellTracker CM-DiI Dye and Dynal-MPC-L magnetic particle concentrator were obtained from Life Technologies (Grand Island, NY). Fluorenylmethyloxycarbonyl (fmoc) protected amino acids and reagents were purchased from various sources. Fmoc-N-amido-dPEGn-acid was obtained from Quanta BioDesign, Ltd (Plain City, OH). Fmoc-glu(Otbu)-OH, Fmoc-proline-OH, and Kaiser Test kit were purchased from AnaSpec (Fremont, CA). N10-(trifluoroacetyl)pteroic acid was a generous gift from Endocyte Inc. (West Lafayette, IN). All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). KB (an FR + HeLa-derived human cervical cancer line) and MDA-MB-231 cells were purchased from ATCC. Folate-FITC was generous gift from On Target Laboratories Inc. (West Lafayette, IN). Non-small cell lung cancer patient blood samples were provided by Dr. Sunil Singhal, University of Pennsylvania. All animal and human tissue-related experiments were performed in accordance with relevant guidelines and regulations. The studies were carried out in compliance with the ARRIVE guidelines.
3
Visualizing pGFPns Delivery to HSPCs
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To visualize the delivery of pGFPns to HSPCs, at 24 and 72 hours, HSPCs from the coculture with Cy5-pGFPns were examined by ELYRA PS.1 Superresolution Microscopy (Zeiss). Briefly, cells were first stained with CellTracker CM-DiI Dye (Invitrogen) for lipid membrane staining and seeded onto poly-l -lysine–coated coverslips. After 10 min, cells were fixed with 4% paraformaldehyde for 10 min at room temperature. After washing with PBS thrice, samples were mounted with SlowFade Diamond Antifade Mountant with DAPI (Invitrogen) for imaging.
4
Myocardial Infarction Mouse Model Study
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All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of The Catholic University of Korea (Approval number: CUMC-2015-0048-04). Myocardial infarction in the rodent model was induced as we previously described [28 (link),29 (link),30 (link)]. Male BALB/c nude mouse (8-week-old, 30–40 g; Orient bio, Seongnam, Korea) anesthetized via inhalation of 2% isoflurane were intubated through the trachea with an 18 G intravenous catheter. Then, the mice were mechanically ventilated with medical-grade oxygen. After the left thoracotomy was performed, MI was induced by the left anterior descending artery’s permanent ligation. Immediately after MI induction, BM-MSC cells, HGF-eMSC cells, or a mixture (BM-MSC and HGF-eMSC) of cells were injected directly into the ischemic area. To trace the BM-MSCs within the heart tissues further, the BM-MSCs were pre-labeled with a red fluorescence dye, CM-DiI, prior to cell injection. Briefly, BM-MSCs were stained using CellTracker™ CM-DiI Dye (Invitrogen™, Carlsbad, CA, USA). BM-MSCs were incubated in the CM-DiI Dye solution (5 µg/mL) for 5 min at 37 °C, followed by additional 15 min at 4 °C. After labeling, BM-MSCs were washed with phosphate-buffered saline (PBS) and re-suspend in serum free medium for further uses.
5
Detailed Multidisciplinary Research Protocol
6
Isolation and Culture of Human Hair Follicle Cells
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Healthy young Koreans, with no obvious scalp diseases, provided occipital scalp skin tissue for this study. From the tissue, hHFs were isolated and dissected into single follicular units with a No. 20 blade under a stereomicroscope (Olympus). The micro-dissected hHFs were used for primary hDP and hORS cell cultures. The hDP cells were cultured as described previously (Magerl et al., 2002 (link)). In short, candlelight-shaped dermal papilla (DP) of HFs were dissociated in Dulbecco’s Modified Eagle’s Medium (Welgene) supplemented with 10% fetal bovine serum (Welgene) and antibiotic/antimycotic solution (penicillin and streptomycin; Gibco). For hORS cells, the hair shaft and bulb region of the dissected hHF were cut off and immersed in DMEM supplemented with 20% FBS, as described by Choi et al. (2019) . On the third day, the medium was changed to Epilife medium (Gibco) supplemented with human keratinocyte growth supplement (Gibco) and antibiotics/antimycotics. We used a fluorochrome (Cell Tracker CM-DiI Dye or Qtracker 525 Cell Labeling Kit, Invitrogen), in accordance with the manufacturer’s instruction, to label the cultured hORS or hDP cells, respectively. All cells were kept in a humidified 5% CO2 atmosphere at 37°C.
7
Transwell Invasion and Transendothelial Assay
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Transwell Matrigel invasion assays and transendothelial invasion experiments were conducted with 24‐well transwell inserts containing a polycarbonate membrane (8‐μm pore size; Corning). For the transendothelial invasion experiment, 1 × 105 HUVEC cells were seeded into transwell plates before being coated with Corning Matrigel matrix (Corning, Cat. #: 354234) for 24 h. Then, the cells were stained with CellTracker™ CM‐DiI Dye (Invitrogen, Cat. #: C7000) following the manufacturer's instructions and added into the upper chambers. 500 μl mediumwith 20% FBS was then added into the lower chambers. The plates were maintained at 37°C for 15 h. The invading cells were then fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Cells were then counted using a Zeiss Axio Imager.Z2 microscope.
8
CM-DiI Dye Microinjection Into LPM
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CellTracker CM-DiI Dye (C7000; Invitrogen) was microinjected using filamented glass capillaries at a concentration of 1 µg µl−1 into the LPM, just posterior to Kupffer’s vesicle at the approximately four-somites stage using Eppendorf FemtoJet.
9
Immunohistochemical Visualization of Tracheal Mucin
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Thoracic tracheal tubes of each beetle in different treatments were dissected and instantly fixed with 4% paraformaldehyde overnight. After being washed with 1× PBS buffer, the samples were permeabilized with 0.5% TritonX-100/PBS at 37°C for 20 min, then blocked with 5% goat serum for 1 hr, and incubated with affinity-purified polyclonal rabbit antibody against Muc91C (produced by BGI, China, 1: 100) at 4°C for 24 hr. The samples were then washed three times for 5 min each with 0.5% TritonX-100/PBS, Alexa Fluor-488 goat anti-rabbit IgG (Invitrogen, A11008, 1: 500) was used as the secondary antibody. The cellular nucleus was stained with Hoechst 33342 (Invitrogen H3570, 1:1000) for 20 min. The cell membrane system was stained with CellTracker CM-DiI dye (Invitrogen, C7001, 1: 500) for 30 min. Finally, the samples were mounted with Fluoroshield mounting medium (Solarbio, S2100). Fluorescence was detected using an confocal laser-scanning microscope (SP8 Lightning, Leica). Taenidial folds are autofluorescence excitated by 488 nm or 561 nm lasers and have higher autofluorescence excitated by 561 nm laser. Images were processed using LAS X Navigator (Leica).
10
Tracking T Cell Activation via Fluorescence
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HONE1 cells stained with the CellTracker CM-DiI Dye (C7000, Invitrogen) and activated human primary T cells labeled with live-cell fluorescent dye CMFDA (5-chloromethylfluorescein diacetate, C7025, Invitrogen) at the ratio of 1: 10 were cocultured for 3 hours. The Operetta CLS high-content cell imaging analysis system (PerkinElmer) was used to track the fluorescence status of T cells in real time.
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