Isotype control

Staining for cyclins was performed according to the method described by Darzynkiewicz et al^{38 (link)}. Briefly, subconfluent cultures of T98G and MO59K cells were treated with the indicated concentrations karenitecin in the presence and absence of radiation for 72 hours. The cells were harvested and fixed in ice-cold ethanol overnight at 4° C. The cells were washed, centrifuged, and suspended in permeabilization solution (0.25%v/v Triton X-100 in PBS pH 7.4) and kept on ice for 5 minutes. The cells were washed again, resuspended in 100μl wash buffer (1% BSA in PBS); and incubated with antibodies to cyclins B, D or isotype control (Santa Cruz biotechnology, CA) for 60 minutes. The cells were washed, and stained with goat anti-mouse FITC for 30 minutes at RT. The stained cell preparation was stained with 5 μg/ml PI and 200 μg/ml DNase-free RNase A. The cells were analyzed on a FACScan flow cytometer (Becton Dickinson, San Jose, CA) and the results were acquired using CELL quest software and the Modfit LT version 2.0 (Verity Software, Topham, Maine). PI staining was used to reveal the various phases of the cell cycle and the cyclin staining in the G₀/G₁ and G₂/M phase of the cell cycle was estimated as the MFI after subtracting the Mean Fluorescence Intensity recorded by the isotype control for each sample.

CTR LSECs were plated on fibronectin-coated in 10% FBS-DMEM, at 37°C/5%CO₂. Non-adherent cells were removed and LSECs were incubated for 15 min with FBS-free DMEM, anti-human CD36 Antibody (0.01μg/mL - STEMCELL™ Technologies, cat#60084) or Isotype control (0.01μg/mL - Santa Cruz Biotechnology, Cat# sc-3877). Then, cells were and harvested for cGMP measurement.