Method for preparing cancer stem cell spheroids
A technology of cancer stem cells and cancer cells, which is applied in the field of drugs for the treatment of cancer cell resistance, and can solve problems such as insufficient inclusion of cancer stem cells
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example 1
[0101] Reference Example 1: Analysis of Heterogeneous Tumor Formation
[0102] Female BALB / c nude mice (6 weeks) were obtained from Orient Bio Inc. and stored under sterile conditions in the animal laboratory of the Korea Advanced Institute of Science and Technology middle. Mice were randomly assigned to randomized experimental groups. All operations were performed under isoflurane anesthesia, and for ethical procedures and scientific management, all animal-related procedures were reviewed and approved by the Korea Advanced Institute of Science and Technology, Institutional Animal Care and Use Committee (KAIST-IACUC) (approval number: KA2014- twenty one).
[0103] In addition, in order to prepare a heterogeneous model of human ovarian cancer, different series of concentrations (10 6 to 10 2 2D cultured control SKOV3 cells) or SKOV3-ssiCSCs isolated from their corresponding spheroids were mixed with 50% Matrigel (Corning), and then subcutaneously injected into 6-week-old fe...
example 3
[0106] Reference Example 3: Histological Analysis and Immunohistochemistry
[0107] Liver biopsy samples obtained from BALB / c nude mice inoculated with 2D control or SKOV3-ssiCSC cancer cells were fixed with 10% formalin, dehydrated and embedded in paraffin, cut into 5 μm thick samples, and placed in on a glass slide. Samples were deparaffinized and stained with hematoxylin % eosin (H&E) for histological evaluation with a standard light microscope (Eclipse 80i, Nickon).
[0108] Liver metastases were confirmed by immunohistochemistry after paraffin embedding and fragmentation (5 μm) of the tissue. The broken liver tissue was sterilized with 10 mM sodium citrate buffer (pH 6.0) for antigen retrieval, blocked with PBS containing 5% bovine serum albumin (BSA) and 1% goat serum, and then incubated with rabbit antibody Human TNC primary antibody (20 μg / mL; cat# AB19011; Millipore) was incubated for 1 hour at room temperature (RT). After incubation, slides were washed with D-PBS, i...
example 4
[0109] Reference Example 4: Western Blot Analysis
[0110] 2D control SKOV3 cells and SKOV3-ssiCSC spheroids were lysed with RIPA lysis buffer (ThermoFisher Scientific) containing protease inhibition cocktail for 30 min on ice. Lysate protein was quantified using the Bradford protein assay kit (Bio-Rad), and equal amounts of protein (50 μg) were separated by electrophoresis using a Bolt 4-12% Bis-Tris Plus polyacrylamide gel (ThermoFisher Scientific). Gels were dry-blotted onto PVDF (polyvinylidene difluoride) membranes using the iBlot2 transfer system (ThermoFisher Scientific) according to the manufacturer's instructions.
[0111] PVDF membrane was passed through the primary antibody of rabbit anti-phospho-β-catenin (1:1000, catalog number 9561; Cell Signaling Technology), mouse anti-β-catenin antibody (1:1000, catalog number 13-8400; Invitrogen) and rabbit anti- Western blots were incubated with GAPDH antibody (1:1000, cat. #25778; Santa Cruz Biotechnology), which was then ...
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