Human breast cancer (MCF7, MDA-MB-231, T47D, and SK-BR-3) cells were purchased from the Korean Cell Line Bank (Seoul, Korea). Normal human breast (MCF10A) cells and primary human umbilical vein endothelial cells (HUVECs) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). MCF7, T47D, and SK-BR-3 cells were grown in the RPMI-1640 medium (Gibco, Grand Island, NY, USA), and MDA-MB-231 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) containing 10% fetal bovine serum (FBS, Sigma, Darmstadt, Germany) with 100 mg/mL penicillin and streptomycin (P/S, GenDEPOT, Barker, TX, USA). MCF10A cells were grown in the mammary epithelial basal medium (CC-3151; Lonza, Basel, Switzerland) containing supplements (CC-4136; Lonza). HUVECs were grown in endothelial basal medium-2 (CC-3156; Lonza) containing supplements (CC-4176; Lonza) according to the manufacturer’s instructions.
Cc 4176
Manufactured by Lonza
Sourced in Switzerland
The CC-4176 is a specialized laboratory instrument designed for cell culture applications. It serves as a controlled environment for the cultivation and maintenance of cells. The core function of the CC-4176 is to provide a stable and regulated atmosphere for cell growth and development.
Automatically generated - may contain errors
Lab products found in correlation
Cc 3156, by Lonza (12 mentions)
Huvecs, by Lonza (5 mentions)
Cc 3162, by Lonza (5 mentions)
Ebm 2, by Lonza (3 mentions)
Cc 2519, by Lonza (2 mentions)
Egm 2 bulletkit, by Lonza (2 mentions)
Trypsin edta, by Thermo Fisher Scientific (2 mentions)
Metamorph, by Molecular Devices (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Mda mb 231, by Thermo Fisher Scientific (1 mentions)
Sk br 3, by Thermo Fisher Scientific (1 mentions)
Huvecs, by American Type Culture Collection (1 mentions)
Mcf 7, by Thermo Fisher Scientific (1 mentions)
Sk br 3, by Korean Cell Line Bank (1 mentions)
Mcf 7, by Korean Cell Line Bank (1 mentions)
Mda mb 231, by Korean Cell Line Bank (1 mentions)
Cc 3151, by Lonza (1 mentions)
Cc 4136, by Lonza (1 mentions)
Egm 2 bulletkit medium, by Lonza (1 mentions)
24 protocols using cc 4176
1
Human Breast Cancer Cell Lines Protocol
2
Culturing and Cryopreserving HUVECs
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Primary Human Umbilical Vein Endothelial Cells (HUVECs) were purchased from Lonza (C2519AS) and used up to passage five. Early passage HUVECs were cultured in EGM™2-Bulletkit™ medium with growth supplements CC-3156 & CC-4176 purchased from Lonza. At 80% confluency, HUVECs were trypsinized, washed twice with phosphate-buffered saline (PBS), pelleted, and frozen at −80°C.
3
VEGFA-Induced Endothelial Cell Responses
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For routine culture, HUVECs (CC-2519, Lonza) and human pulmonary artery endothelial cell (HPAEC, CC-2530, Lonza) between three to seven passages were maintained in endothelial growth medium medium 2 (EGM2, cc-3162, Lonza) with all the growth factor supplied. For VEGFA stimulation experiments, HUVECs were cultured overnight in basal endothelial cell growth medium 2 medium (EBM2, CC-4176 Lonza) with 1% fetal bovine serum (FBS). A total amount of 50 ng/ml VEGFA was then added, and cells were collected at 0, 1, 4, and 12 h for ChIP-qPCR, ChIP-seq, RNA-seq, and RT-qPCR. Where indicated, flavopiridol (100 nM), JQ1 (500 nM), or DZNep (500 nM) were added 1 h before VEGFA stimulation.
Human MSCs (hMSCs) were purchased from Lonza (Catalog #: PT-2501) and cultured in MSCBMTM MSC basal medium (PT-3238, Lonza).
siRNAs were synthesized at GenePharma with all sequences listed in the Supplementary Table3 . A total amount of 10 ng/ml siRNA were transfected with Lipofectamine® RNAiMAX regent manufactured by Thermo Fisher Scientific. The serum starvation was performed one day after siRNA transfection.
Human MSCs (hMSCs) were purchased from Lonza (Catalog #: PT-2501) and cultured in MSCBMTM MSC basal medium (PT-3238, Lonza).
siRNAs were synthesized at GenePharma with all sequences listed in the Supplementary Table
4
Pancreatic Cancer Cell Lines Protocol
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Pancreatic ductal adenocarcinoma cell lines, AsPC-1, BxPC3, PANC-1 and MIA PaCa-2 (ATCC), were maintained in RPMI 1640 media containing 10% foetal bovine serum (FBS), glutamineand sodium pyruvate. To collect conditioned media, cells were treated with serum-free RPMI 1640 media, after 24 h, the conditioned media was filtered and stored for subsequent uses. LECs were purchased (PromoCell) and cultured in endothelial cell growth medium MV2 with supplement (PromoCell). HUVECs were cultured in EGM-2/M199/20% FBS (Lonza, EGM-2 BulletKit, CC-3156 and CC-4176). Cell lines used in this study have been authenticated and tested as mycoplasma free. Stable knockdown of DUSP2 was achieved in PANC-1 cells using two different shRNAs (clone TRCN0000355667 and TRCN0000355666, RNAi core lab of Genomics Research Center, Academia Sinica, Taipei, Taiwan) and lentivirus delivery according to manufacturer’s protocol. Stable cells were selected with puromycin (1 μg/ml).
5
Culturing Human Umbilical Cord Endothelial Cells
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Human umbilical cord endothelial cells (HUVEC) were obtained from the cell culture Depository of Live Systems (Lomonosov Moscow State University), ID of the collection MSU_HUVEC. HUVEC were cultured in EGM™ Media containing 2% fetal bovine serum (FBS) and recombinant growth factors (CC-4176, Lonza) at 37 °C in a 5% CO2 incubator. Cells were passaged at 70% confluency using HyQTase solution (HyClone). For migration assay, cells were used between passage 3 and 6.
6
Isolation and Culture of Mouse SCMECs
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SCMECs were isolated from the spinal cord of 8-week-old mice. After euthanizing the mice, the entire spine was take out sterility. Inject cold PBS (Solarbio, P1020) through the sacral opening of the spine to expel the entire spinal cord from the spine. After removing the dura mater and cutting the spinal cord tissue, the tissue was digested using 0.1% collagenase II (Gibco, 17101015). Centrifuge at 4000 rpm in 20% BSA (Meilunbio, MB4219) for myelin removal. Next, digest the tissue with 0.1% collagenase/dispersase (Roche, 10269638001). After washing with complete culture medium (EGM-2, Lonza, CC-4176), pellets were re-suspended in complete culture medium with 5% FBS (Opcel, BS-1105) and plated onto T25 flask coated with collagen I rat tail (Gibco,10483-01) for further experiments.
7
Culturing Human Endothelial Cells
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HUVECs were purchased from Gibco. HUAECs were from Provitro. HUVECs and HUAECs were cultured in EGM-2 medium (CC-3156, Lonza) containing supplements (CC-4176 or CC-4147), respectively, and were used for a maximal number of 6 passages. All cells were cultured at 37 °C and 5% CO2 and were tested negative for mycoplasma contamination before experiments.
8
Evaluating pNaKtide effects on retinal cells
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Human retinal microvascular endothelial cells (HRMECs) and human retinal pigment epithelium (ARPE-19) cells were purchased from Cell Systems (CSC 2M1; Kirkland, WA, USA) and American Type Culture Collection (CRL-2302; Manassas, VA, USA), respectively. EGM-2 Medium and HyClone Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM-F12) were purchased from Lonza (CC-3162; Basel, Switzerland) and Cytiva (SH30023.01; Marlborough, MA, USA), respectively. HRMECs were cultured in EGM-2 Medium supplemented with growth factors (CC-4176, Lonza), 5% fetal bovine serum, 100 units/mL penicillin, and 100 µg/mL streptomycin. ARPE-19 cells were cultured in DMEM-F12 media supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 µg/mL streptomycin. Cells were kept at 37°C in humidified air with 5% CO2. HRMECs and ARPE-19 cells were treated with or without pNaKtide or a scrambled peptide with a Tat leader sequence (Tat-scrambled peptide, sequences listed in Supplementary Table S1 ), at a concentration of 0.5, 1, or 5 µM for 24 hours. A Trypan blue exclusion assay was performed to evaluate cell viability.
9
HUVEC Proliferation and Preparation
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Human umbilical vein endothelial cells (HUVEC) from pooled donors were purchased at passage zero (Lonza, Basel, Switzerland; #CC-2519) and used for experiments in passages 2–5. HUVECs proliferated on 10cm collagen coated plastic cell culture plates to 100% confluence in EGM-2 media containing 2% fetal bovine serum (FBS) (Lonza, Basel, Switzerland; #CC-3162 & CC-4176) in humidified chambers at 37°C, 5% CO2. Once cells reached confluence, they were transferred to 96-well collagen coated plates for treatments and subsequent experiments.
10
Immunostaining of Angiogenic Markers in HUVECs
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Human umbilical vein endothelial cells, HUVECs were obtained from ATCC and maintained in Endothelial Growth Media, EGM-2 Bulletkit (CC-3162 and CC-4176, Lonza). Similar number of cells were grown on coverslips coated with matrigel in a six-well dish for four hours. Upon formation of capillary tubes, cells were fixed with 4% paraformaldehyde for 20 min at room temperature. The fixed cells were then permeabilized with 0.1% TritonX-100/PBS for 10 min, followed by blocking with 4% BSA/PBS 1 h at room temperature. Primary antibodies were 1:100 diluted in 1% TritonX-100/PBS and incubated with the cells at 4 °C overnight. Secondary antibodies were diluted 1:1000 in 1% TritonX-100/PBS with 1 h incubation at room temperature. Images were taken at 20× and 40× magnification with an Axio observer Z1 and PerkinElmer spinning disk confocal microscope. All immuno-staining were conducted with at least three independent experiments. The antibodies used were anti-CD34 (GeneTex and Abcam), Notch 1 (Santa Cruz), Dll4 (Abcam).
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