One day before the assay, NK cells were grown in culture in NK cell media supplemented with IL-2 (50 IU/mL) or IL-15 (10 ng/mL) for 12 to 18 hours, and neuroblastoma cells were plated at a density of 50,000 cells per well in 4-well glass bottom chamber slides (Ibidi). On the day of the assay, neuroblastoma cells were labeled with calcein-AM, and NK cells were labeled with CellTracker Orange (Thermo Fisher Scientific), according to manufacturer instructions. Hu14.18K322A (10 μg/mL) was added to the wells for 1 hour to allow binding to tumor cells. Dye-labeled NK cells were then added to the wells containing neuroblastoma cells. A C2 Nikon confocal microscope (Nikon) was used to perform live-cell imaging at a magnitude of 10X for 12 hours. Five different areas were imaged per condition. To quantify the imaging data, we extracted the green (8 bit) and red (16 bit) channels and segmented the green and red signal with FIJI software (12 (link)) and Weka Trainable Segmentation plug-in (13 ). We converted the segmented images into a binary mask and applied the 3D ROI manager plug-in to calculate contact area in μm and signal intensity (14 (link)).
4 well glass bottom chamber slides
Manufactured by Ibidi
Sourced in Germany
The 4-well glass bottom chamber slides provide a multi-well platform with a transparent glass bottom for microscopic observation of cell cultures and other biological samples. The slides are designed for convenient and reliable in vitro cell studies.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using 4 well glass bottom chamber slides
1
Live-cell Imaging of NK-Neuroblastoma Interaction
2
Culturing MCF7 Breast Cancer Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All reagents used for cell culturing were purchased from Thermofisher Scientific (Gibco™). Michigan Cancer Foundation-7 (MCF7) cells were originally obtained from The European Collection of Authenticated Cell Culture and were stored in liquid nitrogen until cultured. The cells were cultured in a 5% CO2 in air, 37 °C humidified incubator using phenol red-free low glucose (1 mg/mL) Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% foetal bovine serum (FBS), 2 mM glutamine and 1% penicillin/streptomycin and passaged every 2 days. For imaging experiments, cells were seeded at a density of 1 × 105 cells/ml per well in 4-well glass bottom chamber slides (Ibidi, Germany) or a 3 ml glass bottom dish (Ibidi, Germany) and incubated for 24 h before imaging.
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!