Endometrial adenocarcinoma samples from 70 patients were assembled into tissue microarrays. These patients had received neither prior medical treatment nor radiotherapy for cancer. The normal endometrial, simple hyperplasia endometrial, complex hyperplasia endometrial, and atypical hyperplasia endometrial samples were from 22, 23, 21, and 26 patients, respectively. These samples were prepared from paraffin blocks obtained from the Pathology Department at the Beijing Chao-Yang Hospital of Capital Medical University. The patients' clinicopathological data were retrospectively achieved from the Department of Obstetrics and Gynecology of Beijing Chao-Yang Hospital. The endometrial adenocarcinoma cell line was the Ishikawa cell line purchased from the American Type Culture Collection (Manassas, VA, USA).
Ishikawa cell line
Manufactured by American Type Culture Collection
Sourced in United States
The Ishikawa cell line is a well-established in vitro model derived from human endometrial adenocarcinoma. It is commonly used in research related to endometrial physiology and pathology.
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Lab products found in correlation
10 cm dishes, by Corning (1 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (1 mentions)
L glutamine, by Quality Biological (1 mentions)
Penicillin streptavidin, by Thermo Fisher Scientific (1 mentions)
Tgf β1, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Ozyme (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Merck Group (1 mentions)
5 protocols using ishikawa cell line
1
Endometrial Adenocarcinoma Tissue Microarray
2
Establishment and Characterization of Endometrial Adenocarcinoma Cell Lines
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The human endometrial adenocarcinoma cell line, the Ishikawa cell line [92 (link)], was obtained from American Type Culture Collection (ATCC) (Manassas, VA) [93 (link)]. The primary tumor-derived endometrial adenocarcinoma cell lines originated from patients with Stage IC Grade 3 (ACI-181), Stage IIB Grade 2 (ACI-52), and Stage IIIC Grade 2 (ACI-80) International Federation of Gynecology and Obstetrics (FIGO) staging (gift from Dr. Risinger, Michigan State University, Department of Obstetrics, Gynecology and Reproductive Biology, Michigan State University, Grand Rapids 49503, MI, USA) (Supplementary Table 1). All cell lines used in this study are identified as having endometrioid histologic characteristics and were grown-maintained as previously described [74 (link), 75 (link)]. In brief, cells were grown in phenol red free-DMEM (Cellgro-Mediatech, Inc., Manassas, VA, USA) supplemented with 10% FBS (Atlanta Biologicals, Lawrenceville, GA, USA) and 2 mM L-Glutamine (Quality Biological Inc., Gaithersburg, MD, USA). These cells were maintained at 37°C with humidified atmosphere of 5% CO2 until 90–100% confluence was reached. Cells were subsequently passaged in a 1:5 ratio into 10-cm dishes (Costar, Corning, NY, USA).
3
Cell Culture Protocol for EMT Induction
4
CRKL Overexpression in Ishikawa Cells
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The Ishikawa cell line was obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in Dulbecco's modified Eagle medium (Invitrogen; Thermo Fisher Scientific, Inc.) containing 10% fetal calf serum (Invitrogen; Thermo Fisher Scientific, Inc.), 100 IU/ml penicillin (Sigma-Aldrich) and 100 µg/ml streptomycin (Sigma-Aldrich) at 37°C in an atmosphere of 5% CO2. Cells were grown on sterilized culture dishes and were passaged every 2 days with 0.25% trypsin (Invitrogen; Thermo Fisher Scientific, Inc.).
The pCMV6-CRKL plasmid was purchased from OriGene Technologies, Inc. (Rockville, MD, USA), and was transfected into the cells using Lipofectamine 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The pCMV6 empty vector was used as a negative control. Cells were harvested 48 h after transfection and subjected to various analyses.
The pCMV6-CRKL plasmid was purchased from OriGene Technologies, Inc. (Rockville, MD, USA), and was transfected into the cells using Lipofectamine 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The pCMV6 empty vector was used as a negative control. Cells were harvested 48 h after transfection and subjected to various analyses.
5
Endometrial Stromal Cell Isolation and Ishikawa Cell Culture
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Endometrial tissues were cut into 2–3 mm pieces and digested with 1 mg/mL of collagenase type 1A in DMEM/F12 for 1.5 h at 37°C. It was then filtered through 250 µm nylon sieve to remove mucus and undigested tissue. The filter was passed through a 40 µm nylon sieve, which allowed the stromal cells to pass through, while intact glands were retained. Glands were recovered from the filter by backwashing with DMEM/F12 containing 10% bovine serum albumin and seeded on to the six-well plates for future study for certain durations.
The Ishikawa cell line (American Type Culture Collection, Manassas, VA, USA) was obtained from Shanghai Institutes for Biological Science and maintained in RPMI-1640 medium (Gibco; Thermo Fisher Scientific) containing 10% fetal bovine serum (FBS) and 100 U/mL penicillin and streptomycin antibiotics. When the cells reached confluence, the medium was replaced with phenol red free RPMI-1640 supplemented with 10% charcoal/dextran-treated FBS (all purchased from Sigma-Aldrich). For hormonal treatments, E2 (Sigma-Aldrich) was added to the culture media to a final concentration of 10−9 M (close to the physiological concentration in women at the mid-secretory phase) and 10−7 M (close to the supraphysiological concentration in women with controlled ovarian hyperstimulation), for certain durations according to the experimental purposes (Valbuena et al. 2001 (link)).
The Ishikawa cell line (American Type Culture Collection, Manassas, VA, USA) was obtained from Shanghai Institutes for Biological Science and maintained in RPMI-1640 medium (Gibco; Thermo Fisher Scientific) containing 10% fetal bovine serum (FBS) and 100 U/mL penicillin and streptomycin antibiotics. When the cells reached confluence, the medium was replaced with phenol red free RPMI-1640 supplemented with 10% charcoal/dextran-treated FBS (all purchased from Sigma-Aldrich). For hormonal treatments, E2 (Sigma-Aldrich) was added to the culture media to a final concentration of 10−9 M (close to the physiological concentration in women at the mid-secretory phase) and 10−7 M (close to the supraphysiological concentration in women with controlled ovarian hyperstimulation), for certain durations according to the experimental purposes (Valbuena et al. 2001 (link)).
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