Human cancer cell lines NCCIT, NTERA-2 a1.D1, JAR, JEG-3, and SKBR3 were obtained from ATCC (CRL-2073, CRL-1973, HTB-144, HTB-36, and HTB-20). A549 and HeLa were obtained from RIKEN (RBRC-RCB0098, RBRC-RCB0007). PANC-1 was obtained from DS pharma biomedical (EC87092802-F0). All samples used were tested for mycoplasma contamination. We confirmed that our cell lines are negative to mycoplasma contamination. The piggyBac transposon vector carrying tetO-HA-Dmrt1/Venus-ires-mCherry-Ef1a-rtTA and pCAG-PBase was transfected into human cancer cell lines using Lipofectamine 2000 (Thermo Fisher Scientific). After antibiotic selection with 350 μg/mL G418, the surviving cells were expanded and used for analyses.
Htb 144
Manufactured by American Type Culture Collection
Sourced in United States
The HTB-144 is a laboratory equipment product that serves as a cell culture incubator. It is designed to maintain optimal environmental conditions for the growth and maintenance of cell cultures.
Automatically generated - may contain errors
Lab products found in correlation
Jeg 3, by American Type Culture Collection (2 mentions)
Htb 36, by American Type Culture Collection (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Skbr3, by American Type Culture Collection (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Htb 20, by American Type Culture Collection (1 mentions)
Crl 1973, by American Type Culture Collection (1 mentions)
17β estradiol, by Merck Group (1 mentions)
Crl 1573, by American Type Culture Collection (1 mentions)
Dulbecco modified eagle medium (dmem), by Corning (1 mentions)
Minimum essential medium (mem), by Corning (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
1640 media, by Thermo Fisher Scientific (1 mentions)
8 protocols using htb 144
1
Genetic engineering of human cancer cells
2
Culturing Human Choriocarcinoma and Trophoblast Cell Lines
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BeWo human choriocarcinoma cells (CCL-98, American Type Culture Collection (ATCC) and the immortalized human first-trimester extravillous trophoblast cell line Swan-71 (Straszewski-Chavez et al. 2009 (link)) were maintained in 1:1 DMEM:Ham’s F-12 supplemented with 10% FBS, penicillin (0.1 U/mL), streptomycin (100 μg/mL) and l -glutamine (2 mM) at 37°C, 5% CO2. JAR (ATCC. HTB-144) and JEG-3 (ATCC 92120308) choriocarcinoma cells were cultured in DMEM, containing 10% FBS and penicillin (0.1 U/mL), streptomycin (100 μg/mL)), 2 mM glutamine at 37°C, 5% CO2.
3
In Vitro Trophoblast Adhesion Assay
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JAr cells (JAr, HTB-144, ATCC, Manassas, VA, USA) are a trophoblastic tumor cell line of placental origin that express the placental hormone and differentiate into syncytiotrophoblasts. We used multicellular spheroids of human choriocarcinoma JAr cells as an in vitro attachment model as previously described [11 (link)]. We treated OE-E6/E7 cells at 40–50% confluency with 10 nmol/L 17β-estradiol (Sigma-Aldrich, St Louis, MO, USA) and 1 nmol/L P4 (Sigma-Aldrich, St Louis, MO, USA) to mimic the hormonal environment under normal physiological conditions. The cells were treated with LNG and P4 at various concentrations until full confluency was reached. At 80–90% confluency, the OE-E6/E7 cells were treated with various concentrations of LNG and P4 for 24 h, and JAr spheroids were transferred onto the surface of a confluent monolayer of OE-E6/E7 cells. The cultures were maintained in the culture medium for 6 h. Non-adherent spheroids were removed by centrifugation of the cell culture plates with the cell surface facing down at 15 g for 10 min. We counted the attached spheroids under a light microscope, and the results are expressed as percentages of the total number of seeded spheroids (% adhesion).
4
Cell Culturing of Human Cell Lines
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The human embryonic kidney HEK (ATCC, CRL-1573) cells and human gestational choriocarcinoma JAR cells (ATCC, HTB-144) were purchased from the ATCC. These cells were grown in Dulbecco’s modified Eagle medium (DMEM)-F12; human immortalized chorionic villi cells of first-trimester placenta (HTR-8/SVneo) were grown in Roswell Park Memorial Institute (RPMI)-1640 medium, medium supplemented with 10% fetal bovine serum at 37°C in a humidified atmosphere at 5% CO2. The mycoplasma contamination is examined regularly by immunofluorescence microscopy with DAPI staining according to the guidelines.
5
Culturing Placental Trophoblast-like Cells
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HTR-8/SVneo (HTR-8) (CRL-3271), normal human immortalized first-trimester placental trophoblast-like cells, and choriocarcinoma-derived third-trimester placental trophoblast-like cells (JEG-3 and JAR: referred as trophoblasts) (HTB-36 & HTB-144 respectively) were used and obtained from ATCC. HTR-8 and JAR were cultured in DMEM (Corning) supplemented with 10% fetal bovine serum (Gibco) and antibiotics. JEG-3 cells were maintained in MEM (Corning) supplemented with 10% fetal bovine serum (Gibco) and antibiotics. Cells were maintained at 370 C in a 5% CO2 humidified incubator and passaged regularly (usually 3-4 days). These trophoblast-like cells were referred as trophoblasts in rest of the manuscript.
6
Transfection of Endometrial and Placental Cell Lines
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The human endometrial epithelial RL95-2 (CRL 1671; ATCC, Manassas, VA) and human placenta choriocarcinoma JAR (HTB 144; ATCC) cell lines were maintained in medium supplemented with 10% fetal bovine serum, 100 mg/mL penicillin, and 100 mg/mL streptomycin in 5% CO 2 at 37 C. For transient transfection, the cells were seeded in 6-well culture dishes and transfected with siRNA-CAPS using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA), according to the manufacturer's protocol.
7
Culturing Human Choriocarcinoma Cells for EV Isolation
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The human choriocarcinoma
cell line (JAr) from the first trimester trophoblasts was acquired
from the American Type Culture Collection (ATCC, HTB-144, Teddington,
U.K.). JAr cells were cultured in a T75 flask in Roswell Park Memorial
Institute (RPMI) 1640 media (Gibco, Scotland) supplemented with 1%
penicillin/streptomycin (P/S, Gibco 15140122, Bleiswijk, Netherlands),
1%l -glutamine (Sigma, 59202C, St. Louis), and 10% fetal
bovine serum (FBS, Gibco, 10500064) at 37 °C under moist 5% CO2-rich conditions. At 80% confluence, the conditioned medium
was removed, and the cells were washed with 10 mL of nonsupplemented
RPMI 1640 media to remove traces of FBS. Nonsupplemented RPMI 1640
medium was replaced with fresh RPMI 1640 medium supplemented with
1% penicillin/streptomycin, 1%l -glutamine, and 10% EV-depleted
FBS. Cells were cultured for 24 h at 37 °C and 5% CO2. After incubation, the conditioned medium was collected for EV isolation.
We have submitted all relevant data of our experiments to the EV-TRACK
knowledgebase (EV-TRACK ID: EV190091).43 (link)
cell line (JAr) from the first trimester trophoblasts was acquired
from the American Type Culture Collection (ATCC, HTB-144, Teddington,
U.K.). JAr cells were cultured in a T75 flask in Roswell Park Memorial
Institute (RPMI) 1640 media (Gibco, Scotland) supplemented with 1%
penicillin/streptomycin (P/S, Gibco 15140122, Bleiswijk, Netherlands),
1%
bovine serum (FBS, Gibco, 10500064) at 37 °C under moist 5% CO2-rich conditions. At 80% confluence, the conditioned medium
was removed, and the cells were washed with 10 mL of nonsupplemented
RPMI 1640 media to remove traces of FBS. Nonsupplemented RPMI 1640
medium was replaced with fresh RPMI 1640 medium supplemented with
1% penicillin/streptomycin, 1%
FBS. Cells were cultured for 24 h at 37 °C and 5% CO2. After incubation, the conditioned medium was collected for EV isolation.
We have submitted all relevant data of our experiments to the EV-TRACK
knowledgebase (EV-TRACK ID: EV190091).43 (link)
8
In vitro Spheroid Attachment Assay
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In in vitro attachment model, multicellular spheroids of human choriocarcinoma (JAr) cells (American Type Culture Collection, Manassas, VA, USA; HTB 144) were applied to receptive-phase human endometrial epithelial cell monolayers and Ishikawa cell layers, respectively. The endometrial epithelial cells were pretreated with 10−9 M or 10−7 M E2 and cultured for 3 days. JAr spheroids were prepared according to a standard procedure (Hohn et al. 2000 (link)) and transferred onto the surface of confluent cell monolayers for 1 h (50 spheroids/dish for primary cultured endometrial cells and 45 spheroids/dish for Ishikawa cells). Non-adherent spheroids were detached by centrifugation (10 g ; 10 min) of the six-well plates with the cell surface facing down. We counted the attached spheroids under a light microscope and the attachment rate was calculated for each well as follows: attachment rate equals the ratio of the number of spheroids attached to the number of spheroids seeded. This experiment was repeated at least three times.
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