Murine MT-RET (RET) melanoma cell line was established by isolating and culturing tumor cells from a spontaneously developed tumor in MT-RET transgenic mice (13 (link)). LLC were obtained from ATCC. B16F10-Luc2 cells were purchased from Caliper life sciences, U.S.A. All human melanoma cells (SKMEL-173, SKMEL-28, C-32, WM266-4, A375, M37, SKMEL-147, and SKMEL-23) were kindly provided by J. Utikal. All cancer cells were cultured in DMEM high glucose (Gibco) supplemented with 10% FCS, 1% penicillin/streptomycin (Sigma) and 1x non-essential amino acid (Gibco). Human umbilical vein endothelial cells (HUVEC; Promocell) were cultured in Endopan-3 medium supplemented with growth factors (PAN Biotech GmbH). Mouse lung endothelial cells were acquired from Cell Biologics and were cultured in complete EC media (Cell Biologics). The cell lines used in this study were routinely tested for mycoplasma by PCR. RET and B16F10-Luc2 cells were transduced with lentiviral particles expressing shRNA constructs (Dharmacon): non-targeting (RHS4346), sh-1 Angpt2 (V2LMM_74366) and sh-2 Angpt2 (V2LMM_68229). LLC cells were transduced with lentivirus to overexpress either Angpt2 or control Plenti vector. Control or Angpt2-silenced RET cells were transduced with lentivirus to overexpress either Angpt2 or control Plenti vector for rescuing Angpt2 downregulation.
Complete ec media
Manufactured by Cell Biologics
Complete EC media is a cell culture medium formulated to support the growth and maintenance of endothelial cells. It provides the necessary nutrients and growth factors to facilitate the in vitro culture of endothelial cell lines.
Automatically generated - may contain errors
Lab products found in correlation
Non essential amino acid (neaa), by Thermo Fisher Scientific (1 mentions)
High glucose dmem, by Thermo Fisher Scientific (1 mentions)
B16f10 luc2 cells, by PerkinElmer (1 mentions)
Rhs4346, by Horizon Discovery (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Endopan 3 medium, by PAN Biotech (1 mentions)
Micrococcal nuclease, by Merck Group (1 mentions)
2 protocols using complete ec media
1
Murine Melanoma Cell Lines and Transduction
2
Neutrophil NETosis Modulates Tumor Cell Proliferation
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1 × 105 neutrophils were treated with CAF CMed diluted 1:1 in complete EC media (Cell Biologics) with or without Cl-amidine and incubated for 3 h to generate NETs. The media was then harvested from the NETting or NET inhibited neutrophils. PBS supplemented with 1U/ml micrococcal nuclease (Sigma-Aldrich) was then added to the neutrophils for 10 min at 37 °C and 5% CO2 to detach the NETs. The enzyme was inactivated with 0.5 mM EDTA. PBS containing the NETs was then harvested for xCELLigence assays.
Pancreatic CAF or tumor cells were seeded onto 16 well E-plates (ACEA Biologics) at a density of 5 × 103 cells/well. Cells were allowed to adhere for 1 h. The media was then replaced with the NETting or NET inhibited neutrophil CMed. Plates were placed into an xCELLigence RTCA MP Real-Time Cell Analyzer (ACEA Biologics). Recordings of the impedance, correlating to cell proliferation, were then taken over a 48 h period. The background was then subtracted from the appropriate wells.
Pancreatic CAF or tumor cells were seeded onto 16 well E-plates (ACEA Biologics) at a density of 5 × 103 cells/well. Cells were allowed to adhere for 1 h. The media was then replaced with the NETting or NET inhibited neutrophil CMed. Plates were placed into an xCELLigence RTCA MP Real-Time Cell Analyzer (ACEA Biologics). Recordings of the impedance, correlating to cell proliferation, were then taken over a 48 h period. The background was then subtracted from the appropriate wells.
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