| Assay Method Information | |
| | LAD2 Degranulation Assay (Protocol 2) |
| Description: | LAD-2 cells were maintained at 37oC with 5% CO2 and grown in complete StemPro-34 media supplemented with 1% L-Glutamate, 1% penicillin/streptomycin, and 250 ng/mL human SCF. 18-24 hours prior to experiment, LAD-2 cells were collected and transferred to growth media without SCF. On the day of experiment, cells were collected by centrifugation and seeded in 384-well assay plates, at 4,000 cells per well in 17 ^l freshly made assay buffer (10 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl2, 1.0 mM MgCl2, 5.6 mM glucose, 0.4 mM NaH2PO4, 0.04% BSA, pH = 7.40). For dose-response assays, compounds solubilized in 100% DMSO at 30 mM were serially diluted (1:3) first in 100% DMSO, and 2 intermediate dilutions with assay buffer were used to establish a final top concentration of 3 ^M (in assay plate). For single point assays, dilution of DMSO stocks to 250 nM (in assay plate) was performed in the same manner as described for dose-response assays. Final DMSO concentration was kept constant at 0.1% across plates for all assays. Cortistatin-14 at 30 mM stock concentration was solubilized in HBSS + 0.1% BSA, stored at -20°C in 5 ^L aliquots and discarded after one freeze-thaw cycle. Final concentration of agonist used in both dose-response and single point assays was 0.5 ^M, prepared in assay buffer. Antagonist equilibrium was established with 2 h preincubation, followed by no wash, direct addition, and stimulation of agonist for 30 minutes at 37oC. For detection of ^-hexosaminidase activity, assay plates were centrifuged and 5 ^L of cell-free supernatant from each assay well was transferred to a black-walled black-bottomed 384-well plate. Substrate solution (1 mM 4-Methylumbelliferyl N-acetyl-β-D-glucosaminide in citrate buffer, pH=4.5, 15 ^l) was added to each well and plates were incubated for 1 hour at 37oC. The plate was read on an EnVision XCite plate reader for umbelliferone fluorescence intensity (λex=355 nm, λem=460 nm). Background-subtracted data was analyzed using either GraphPad Prism or a custom Python script to calculate IC50 using either Log[Antagonist] vs. response (4 parameter) nonlinear regression (dose-response assays). |
| Affinity data for this assay | |
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