Monocultures of HUVECs and MSC-HUVEC co-cultures were grown on glass coverslips for 48 h or on MatrigelTM for 19 h. HUVECs were pre-loaded with 5 μM CellTracker ™ Green CMFDA fluorescent dye (ThermoFisher Scientific, Waltham, MA, USA) for 40 min according to the manufacturer’s protocol. Cells were fixed with 4% formaldehyde and probed with the following primary antibodies (Abs): anti-CD31 (Biolegend, San Diego, CA, USA), anti-laminin (Abcam), anti-collagen 1 (Abcam), anti-collagen 4 (Abcam, Cambridge, UK), anti-fibronectin (Abcam, Cambridge, UK), or isotype-matched control immunoglobulins, followed by fluorophore-conjugated secondary Abs: goat anti-rabbit Alexa Fluor 594 (Invitrogen) or goat anti-mouse Alexa Fluor 594 (Invitrogen). Stained cells were visualized using a wide-field fluorescent Axiovert 200 M Microscope (Zeiss, Oberkochen, Germany).
Celltracker green cmfda fluorescent dye
Manufactured by Thermo Fisher Scientific
Sourced in United States
CellTracker™ Green CMFDA is a fluorescent dye used for tracking and visualizing cells. It is a cell-permeant dye that becomes fluorescent upon entering the cell and binding to intracellular components. The dye can be used to label live cells for subsequent monitoring or analysis.
Automatically generated - may contain errors
Lab products found in correlation
Anti laminin, by Abcam (1 mentions)
Anti cd31, by BioLegend (1 mentions)
Anti collagen 4, by Abcam (1 mentions)
Axiovert 200m microscope, by Zeiss (1 mentions)
Anti fibronectin, by Abcam (1 mentions)
Anti collagen 1, by Abcam (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Varioskan flash microplate reader, by Thermo Fisher Scientific (1 mentions)
Alexafluor 647 dextran, by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Merck Group (1 mentions)
Easysep mouse monocyte isolation kit, by STEMCELL (1 mentions)
5 protocols using celltracker green cmfda fluorescent dye
1
HUVEC Monoculture and Co-culture Characterization
2
Trans-endothelial Migration Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For trans-endothelial migration assay, the hCMEC/D3 monolayer was cultured on 3 μm pore filter inserts in phenol red free endothelial medium (described above). On the day of the experiment, TEER of the monolayer was monitored. Chambers with a TEER lower than 35 ohm/cm2 were discarded. Transfected or control BC cells were then labeled using CellTracker™Green CMFDA fluorescent dye (cat# C2925, Thermofischer-Scientific, USA) following the manufacturer’s recommendations. A total of 2 × 104 fluorescent tumor cells were added gently to the hCMEC/D3 monolayer and left to transmigrate to the lower chamber for 24 h at 37 °C. After 24 h, the upper chambers were washed with PBS and scrapped gently with cotton wool. Tumor cells in the basal compartment that have transmigrated through the BEC were lysed using RIPA 1X buffer and fluorescence in the lower compartment was quantified in a fluorescent microplate reader at wavelength excitation/emission: 492/517 nm.
3
Quantifying Breast Cancer Cell Transmigration
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Transmigration of transfected or control MDA231 and MDA231-BrM2 cells was assessed as previously described.13 (link),15 (link) Briefly, pre-transfected MDA231 and MDA231-BrM2 cells were labeled with 5 μM of CellTracker™Green CMFDA fluorescent dye (Thermo Fisher Scientific, USA, cat# C2925) as per the manufacturer’s protocol. Then 5×104 labelled BC cells were gently added to the apical chambers of tight hCMEC/D3 monolayers. BC cells were allowed to transmigrate to the basal compartment containing complete growth medium for 24 hours. The remaining cells on the upper chamber were gently removed using a cotton swab, and BC cells that transmigrated to the basal compartment were lysed with RIPA 1× buffer for fluorescence quantification at a wavelength excitation/emission of 492/517 nm using a Varioskan Flash microplate reader (Thermo Fisher Scientific).
4
Co-culture of MSCs and PBMCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PBMCs were seeded in 12-well plates at a concentration of 2 × 106 cells/well in 2 mL of LGM with the corresponding amount of PHA-L. We increased the amount of cells in this assay to have the possibility to more accurately explore MSCs in co-culture. Upon cultivation in hydrogels or standard 2D conditions, MSCs were labeled with cell permeable CellTracker™ Green CMFDA fluorescent dye (λexcitation 492 nm; λemission 517 nm, Thermo Fisher, USA). For this, cells were incubated in pre-warmed CellTracker working solution at a concentration of 5 µM in standard medium without FCS and incubated for 15 min at 37°C in the dark. Staining was stopped by adding standard medium with FCS and cells were washed two times by centrifugation at 800 × g for 5 min. The number of cells was determined by Trypan blue exclusion staining. CellTracker + MSCs at a concentration of 2 × 105 cells/well were added to PHA-stimulated PBMCs seeded into 12-well plates. After 3 days, cells were collected and stained for flow cytometry as described below.
5
Metastatic Lung Colonization Assays
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For metastatic lung colonization experiments, subconfluent B16F0 monolayers were detached and dissociated to single cells using 0.25% trypsin-EDTA (Sigma-Aldrich) followed by 2X washing in serum-free DMEM. Cells were injected into mice via the tail vein on day 28 in the asthma model, day 21 in the hypersensitivity pneumonitis model, or day 7 after BLM instillation. In select experiments, B16F0 cells were mixed 1:2 with MoDM (sorted from the inflamed lung) or monocytes (isolated from bone marrow using EasySep mouse monocyte isolation kit; STEMCELL Technologies) and injected into the tail vein of uninflamed mice. For late-stage macroscopic lesion assays, mice were injected with 2.5Â10 5 B16F0 cells in 100 mL of DMEM and euthanized 14 days later for the counting of visible pigmented melanoma lesions. For early-stage microscopic lesion assays, 1Â10 6 B16F0 cells expressing EGFP or labeled with the CellTracker Green CMFDA fluorescent dye (5-chloromethylfluorescein diacetate; Thermo Fisher Scientific) were injected into the tail vein of mice in 100 mL of DMEM with or without 5 mg/kg AlexaFluor647-dextran (Molecular Probes) to label the microvasculature. Depending on the assay, the animals were euthanized between 4 and 96 hours after tumor cell inoculation, and the lungs were processed for immunofluorescence or flow cytometry as described below.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!