Co-culturing BC Cells: Culturing Breast Cancer Cells with Bone Fragments to Study Cancer Cell Proliferation

протокол

1. Co-culturing Breast Cancer Cells Adjacent to Bone Fragments to Measure Breast Cell Proliferation Using BLI

1. Prior to the experiment, prepare a suspension containing 2 x 10⁵ breast cancer cells/50 µl, and prepare plugs of bone wax for immobilizing bone fragments in culture. Cut a P200 micropipette tip into 3 pieces and use the narrow end of the largest piece in a “cookie cutter” manner to cut small (~35 mg) pieces of bone wax. Use the wide end of the smallest piece to eject the bone wax plugs into a Petri dish for storage.
2. Place a piece of bone wax at the 12 o’clock position of each well and gently press down with a sterile-gloved index finger.
3. Pipette 50 µl of cell suspension containing 1 x 10⁵ breast cancer cells in DMEM-10% FBS + Pen-Strep in the center of each well of a 6-well plate. (Alternatively, seed cells in a dispersed pattern if desired.) Place the plate in a 37 °C tissue culture incubator for 45 min to promote cell attachment.
4. Place 1 bone fragment onto 1 piece of bone wax for each experimental well and apply gentle pressure with the rongeur to secure it. Perform experiments in triplicate such that 3 bone fragments from a given THR specimen are placed into the top three wells, while the bottom three wells contain bone wax only as controls.
5. Using a 5 ml pipette, gently and slowly deliver 5 ml of DMEM-10% FBS to the side of each well to avoid dislodging the breast cells or bone fragments. Place the plate in a 37 °C tissue culture incubator for 20-24 hr.
6. To perform bioluminescence imaging (BLI) remove the plate from the incubator and add luciferin substrate (to achieve a concentration of 300 µg/ml) to each well. Image the plate immediately on an IVIS Imaging Platform using the following parameters: f-stop of 1, small binning, exposure time of 1 sec, level D. Measure the signal intensity of each well as average radiance (photons/second/square centimeter/steradian).
7. Use imaging software to define Regions of Interest (ROI) to quantitate the average radiance for all experimental and control wells. Export average radiance values to an Excel sheet to perform statistics and generate graphs.

Материалы

Name	Company	Catalog Number	Comments
DMEM (1X) + GlutaMAX-l	Life Technologies	10569-010	Supplement all DMEM with 10% fetal bovine serum and 1% Pen/ Strep
Fetal bovine serum	Life Technologies	16000-044
Penicillin Streptomycin	Life Technologies	15140-122
D-luciferin firefly, potassium salt 5 g (30 mg/ml)	Life Technologies	L-8220
6 Well TC-Treated Cell Culture Cluster	Corning Incorporated	3516
Friedman-Pearson Rongeur	Fine Science Tools	16021-14
Bone wax	Surgical Specialties Corporation	903
IVIS 50 Imaging Platform	Caliper Life Sciences/PerkinElmer
MCF-7 breast cancer cells	ATCC	HTB-22
MDA-MB-231 breast cancer cells	ATCC	HTB-26