Ishikawa cells (well-differentiated human endometrial adenocarcinoma cell) were generously provided by Dr. Bae-Jump, University of North Carolina. HEC-1-A cells (moderately differentiated human adenocarcinoma) were purchased from American Type Cell Culture Collection (ATCC, Manassas, VA). Ishikawa cells were cultured in Minimum Essential Medium Eagle (Sigma, MO, USA) supplied with 1% Non-Essential Amino Acids (NEAA), 5% Fetal Bovine Serum (FBS),100 units/mL penicillin, and 100 μg/mL streptomycin. HEC-1-A cells were cultured in McCoy’s 5A medium (ATCC, Manassas, VA) supplied with 5% FBS, and 100-units/mL penicillin, 100μg/mL streptomycin. Cells were grown and routinely maintained at 37 °C in 10 cm2 culture dishes.
Hec 1a cells
Manufactured by American Type Culture Collection
Sourced in United States
HEC-1A cells are a human endometrial carcinoma cell line. They are adherent, epithelial-like cells derived from the endometrium of a patient with endometrial adenocarcinoma.
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Lab products found in correlation
Minimum essential medium eagle, by Merck Group (1 mentions)
Mccoy s 5a medium, by American Type Culture Collection (1 mentions)
Elisa, by R&D Systems (1 mentions)
Hek293 cell, by American Type Culture Collection (1 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin solution, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (1 mentions)
Penicillin streptomycin solution, by Corning (1 mentions)
Pgl3 basic plasmid, by Promega (1 mentions)
E2758, by Merck Group (1 mentions)
P0130, by Merck Group (1 mentions)
Jar cells, by American Type Culture Collection (1 mentions)
Dmem f12 media, by Thermo Fisher Scientific (1 mentions)
Silencer select sirna, by Thermo Fisher Scientific (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
K562 cell, by American Type Culture Collection (1 mentions)
Yac 1 cells, by American Type Culture Collection (1 mentions)
Biotinylated secondary antibody, by ZSGB-BIO (1 mentions)
11 protocols using hec 1a cells
1
Culturing Ishikawa and HEC-1-A Endometrial Cancer Cells
2
Cytotoxicity and Inflammation Evaluation
3
Evaluating Anti-HSV-2 Activity of Vaginal Swabs
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Using Starplex™ Scientific Starswab II™, CVF was collected from the lateral vaginal wall, frozen and stored at −80°C until processing. Thawed swabs were placed in 300 µl of HEC1A media for 5–10 min, then placed in SpinX insert (MIDSCI, M850003) and centrifuged at 370 g force for 5 min at 4°C to remove all secretions from the swab. To assess the activity of the swab eluent against HSV-2, HEC1A cells (ATCC: The Global Bioresource Center | ATCC) were plated at 200,000/well in a 48 well plate containing McCoy's 5A medium with penicillin/streptomycin. The following day 70 µl of the swab extract were added to the well for a total of 6 h and in the last hour, each well was infected with 200 PFU HSV-2 in 30 µl of media. The treatment/inoculum was removed and 200 µl of fresh media were added. Real-time polymerase chain reaction (PCR) was done on Day 5 using the supernatant to detect HSV-2 DNA and compared to untreated control.
4
Cell Line Cultivation and Maintenance
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HEC1A cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA). Ishikawa cells were kindly provided by Dr. Bruce Lessey (Greenville Health System, SC), and HEC50 cells by Dr. Bryan Hennessy (The University of Texas MD Anderson Cancer Center [MDACC], Houston, TX). Cell lines were maintained at 37°C in a humidified atmosphere of 95% air, 5% CO2 (v/v) in DMEM/F12 (Invitrogen, Carlsbad, CA) supplemented with 10% (v/v) fetal bovine serum (FBS; Atlanta Biologicals, Lawrenceville, GA) and 1% (v/v) penicillin-streptomycin solution (Corning Inc., Corning, NY). HEK293 cells (ATCC) were maintained in RPMI (Invitrogen) supplemented with 5% (v/v) FBS and 1% (v/v) penicillin-streptomycin solution.
5
Endometrial Cancer Cell Lines Protocol
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Human endometrial cancer Ishikawa cells (Ishikawa cells 3H12 No.74) were kindly provided by Dr. Masato Nishida (Kasumigaura Medical Center, Ibaraki, Japan). Human endometrial cancer HEC-1A cells and embryonic kidney 293T cells were obtained from American Type Culture Collection (Rockville, MD, USA). Ishikawa and HEC-1A cells were originally established from a well-differentiated (G1) and moderately differentiated (G2) endometrial adenocarcinoma, respectively [19 (link)]. We confirmed the ERα status of Ishikawa (ERα-positive) and HEC-1A (ERα-negative) cells by qRT-PCR as described elsewhere [20 (link)]. Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, penicillin (100 units/mL), and streptomycin (100 μg/mL) at 37°C in a humidified atmosphere of 5% CO2 in air. HEC-1A cells were transfected with a luciferase expression plasmid, which was generated by inserting a luciferase gene from the pGL3-basic plasmid (Promega, Madison, WI, USA) into the pCXN2 vector [21 (link)], and then stable transformants (HEC-1A-luc) were selected with G-418.
6
Hormone treatment of endometrial cell lines
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HEC-1A cells and JAR cells (American Type Culture Collection, Manassas, VA, USA) were grown in McCoy’s 5 A and RPMI 1640 medium. RL95-2 cells were grown in DMEM/F12 (1:1) with 0.005 mg/mL insulin. The three cells lines were all supplemented with 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin. When the cells reached 80% confluence, they were subsequently starved of serum for 3 h before treatment with P4 (Sigma, P0130), E2 (Sigma, E2758) or E2 and P4 (concentrations can be seen in figure legends) under serum-free conditions. Cells were harvested for RNA after treatment 48 h and for protein after treatment 72 h.
7
Culturing Human Endometrial Cells
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Human endometrial cancer (HEC)-1A cells were purchased from the American Type Culture Collection (ATCC) (Manassa, VA) and maintained in DMEM/F12 medium supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% FBS under standard culture conditions of 37°C, 95% humidified air and 5% CO2. Human endometrial fibroblast cells were isolated using surgical curettages of the hysterectomized uteri of patients at the Ajou University Hospital (Suwon, S. Korea) with no other apparent endometrial disease diagnoses. Informed consent was obtained from patients before surgery according to the Ajou University IRB preapproval (Suwon, S. Korea).
8
BMI-1 Knockdown in HEC1A Cells
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HEC1A cells were obtained from the American Type Culture Collection. Cells were cultured in DMEM:F12 media (Life Technologies) containing 10% (v/v) FBS at 37°C and 5% CO2. Knock‐down experiments were performed using Silencer Select siRNA (ID: S2016) (Ambion®). Cells were seeded in a 6‐well tissue culture plates at a density of 6 × 105 cells/well. To knockdown of BMI‐1, for each well 30 nmol/L control siRNA or siRNA targeting BMI‐1 was complexed to lipofectamine RNAiMAX (Invitrogen™; Thermo Fisher Scientific) following manufacturer's specifications. The effect of BMI‐1 silencing was check after 48 hours.
9
Cell Culture Protocols for NK, Carcinoma, and Lymphoma Cell Lines
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NK-92mi cells (a human IL-2 independent NK cell line) HEC-1A cells (a human endometrial carcinoma cell line), K562 cells (a human lymphoblast cell line), CT-56 cells (a mouse colon carcinoma cell line) and YAC-1 cells (a mouse lymphoma cell line) were obtained from the American Type Culture Collection (Manassas, VA, USA). NK-92mi cells were cultured in α-MEM media supplemented with 100 U/mL penicillin, 100 U/mL streptomycin, 20% FBS (Hyclone, Logan, UT, USA), 1% vitamin solution X100 (Gibco, Waltham, MA, USA) and 2-mercaptoethanol (Gibco). HEC-1A, K562, CT-26 and YAC-1 cells were cultured at 37 °C in DMEM/F12 media supplemented with 100 U/mL penicillin, 100 U/mL streptomycin and 10% FBS in a humidified incubator in the presence of 5% CO2. All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified.
10
Osteopontin Regulates HEC-1A Cell Signaling
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Human uterine epithelial cell line HEC-1A cells were acquired from the American Type Culture Collection (ATCC). Recombinant OPN protein (rhOPN) was purchased from R&D Systems. Rabbit anti-AKT and phosphor-AKT (Ser473) antibodies were purchased from Bioworld Technology. Rabbit anti-ERK, phospho-ERK 1/2, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies and U0126 (a specific inhibitor of MEK/ERK; 10-5M) and LY294002 (a specific inhibitor of p38; 10-6M) were purchased from Beyotime Institute of Biotechnology. MMP-2, E-cadherin, N-cadherin and Vimentin were purchased by Proteintech. Cell Counting Kit-8 was purchased by Dojindo. Enhanced chemiluminescence (ECL) assay kit was purchased from Amersham. Horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody, anti-mouse secondary antibody, biotinylated secondary antibody, streptavidin-horseradish peroxidase and diaminobenzidine (DAB)-peroxidase substrate were purchased from ZSGB-BIO. TRITC-conjugated goat anti-rabbit secondary antibody IgG and FITC-conjugated goat anti-mouse secondary antibody were purchased from Thermo.
Cell lines and culture conditions HEC-1A cells were grown in McCoy's 5A supplement with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin. Cells were maintained at 37 o C under 5% CO 2 in humidified air.
Cell lines and culture conditions HEC-1A cells were grown in McCoy's 5A supplement with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin. Cells were maintained at 37 o C under 5% CO 2 in humidified air.
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