A collagen layer was coated rotating overnight at 4 °C on a clear bottom 96-well black plate (Corning 3603) as previously described [21 (link)]. Cells were then plated in the prepared plate at 50,000 cells per well for a confluent monolayer. Cells were then loaded with 4 μM Fluo4-AM (Life Technologies F14201) and prepared as previously described [21 (link)] prior to the addition of compound. The final volume per well was 100 μL of Hank’s Balanced Salt Solution (HBSS + Ca2+; Gibco 14025-092).
Dilutions of compounds were prepared in a separate plate at 5× concentration for the robotic addition of 25 μL to cells by the FLEX Station 3 Multi-Mode plate reader (Molecular Devices). The FLEX Station 3 reads Relative Fluorescence Units (RFUs) measured every 1.28 s for a period of 300 s after compound addition. The results were either plotted as the maximum-minimum for each compound concentration over the time period or ΔF/F0 as previously calculated [21 (link)] with the initial 30 s reading as a baseline value prior to compound addition. All values shown are mean SD of triplicate samples.
Dilutions of compounds were prepared in a separate plate at 5× concentration for the robotic addition of 25 μL to cells by the FLEX Station 3 Multi-Mode plate reader (Molecular Devices). The FLEX Station 3 reads Relative Fluorescence Units (RFUs) measured every 1.28 s for a period of 300 s after compound addition. The results were either plotted as the maximum-minimum for each compound concentration over the time period or ΔF/F0 as previously calculated [21 (link)] with the initial 30 s reading as a baseline value prior to compound addition. All values shown are mean SD of triplicate samples.
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