CD45-/CD34-/CD31+ Human Lymphatic Endothelial Cell (LEC)
Treatment protocol
Primary LEC were serum starved overnight in EBM supplemented with 0.2% bovine serum albumin. Cells were treated or not for 1h, 4h, 8h or 24h with recombinant human VEGF-A165 (R&D Systems; 20 ng/ml) or mature human VEGF-C (R&D Systems; 500 ng/ml).
Growth protocol
LEC were seeded onto fibronectin-coated culture dishes (10 µg/ml; BD Biosciences, Bedford, MA) and were cultured in endothelial cell basal medium (EBM; Cambrex Bio Science, Walkersville, MD) supplemented with 20% fetal bovine serum (FBS; Invitrogen, Grand Island, NY), 2 mM L glutamine, antibiotic-antimycotic solution, 10 µg/ml hydrocortisone and N6,2'-O-dibutyryl-adenosine 3',5'-cyclic monophosphate (25 µg/ml; all from Fluka, Buchs, Switzerland) for up to 7 passages.
Extracted molecule
total RNA
Extraction protocol
Isolate total cellular RNA with 1 ml of the Trizol reagent (Invitrogen) after washing one 80-90 % confluent 10 cm culture dish with PBS. Homogenize the sample using a cell scraper and add 0.2 ml of chloroform in a 1.5 ml tube. Shake the sample vigorously for 15 seconds and allow standing for 5 minutes. Centrifuge the resulting mixture at 12,000 x g for 15 minutes at 4 degree Celsius. Transfer the aqueous phase to a fresh tube and add 0.5 ml of iso-propanol and mix. Allow the sample to stand for 5 minutes at room temperature and centrifuge at 12,000 x g for 10 minutes at 4 degrees Celsius. Remove the supernatant and wash the RNA pellet by adding 1 ml of 75% ethanol. Vortex the sample and then centrifuge at 12,000 x g for 5 minutes at 4 degrees Celsius. Remove the ethanol and briefly dry the RNA pellet for 5 minutes and add 50 micro-liter of RNAase/DNAase free water and mix by repeated pipetting until RNA pellet is fully dissolved.
Label
Chemiluminescent Digoxigenin-UTP labeled cRNA
Label protocol
Digoxigenin-UTP labeled cRNA was generated from 0.5 µg of total RNA for each sample using Applied Biosystems Chemiluminescent RT-IVT Labeling Kit (P/N 4363105) according to the manufacturer’s protocol.
Hybridization protocol
Hybridization protocol Array hybridization, array processing, chemiluminescence detection, image acquisition, and analysis were performed using Applied Biosystems Chemiluminescence Detection Kit and Applied Biosystems 1700 Chemiluminescent Microarray Analyzer following manufacturer’s protocol.
Scan protocol
Scan protocol Chemiluminescent Detection Kit (P/N 4339627) and Chemiluminescent Microarray Analyzer User Guide (P/N 4338852B).
Description
NA
Data processing
Data processing The Expression Array System Software suite performs the auto-gridding, feature extraction, fluorescence normalization, and signal data generation. The quantification data contain many distinct measurements for each probe, including the three basic measurements: Signal, S/N, and Flag values. The Signal value is the fully corrected, background subtracted measurement of chemiluminescent signal for gene expression values. The S/N value represents the ratio of signal above noise, or the measurement uncertainty of the probe signal, and can be used as a confidence level for the probe measurement. An S/N of 3 represents approximately a 99.95% confidence that the probe is detected above the background noise. In situations where the probe showed S/N < 1, the signal measurement is replaced with a 1 SDEV upper limit based on its probe signal SDEV.
Human dermal lymphatic endothelial cells stimulated with VEGF-A or VEGF-C for 1h, 4h, 8h and 24h
Data table header descriptions
ID_REF
VALUE
VSN normalized log2 transformed signal intensity; S/N ratio: signal to noise ratio generated by the Expression Array System Software; Flag: quality label generated by the Expression Array System Software.
