In the presence of a regular buffer (with calcium), the cytosolic calcium response was even greater indicating that the extracellular calcium may also participate in C7a-induced calcium increase.
Added buffer capacity κD, through loading with the calcium dye, was calculated for the various OGB-1 concentrations using equation 3. The inverse of Δ[Ca2+]AP was plotted against κD in figure 2D in order determine the endogenous buffer capacity κE by back extrapolation to the horizontal axis crossing using the linear relation between (Δ[Ca2+]AP)−1 and κD in equation 4 [12] [12].
Cells were seeded as described earlier and treated with proper inducers and inhibitors and during calcium measurement, cells were trypsinized and calcium dye was used along with the calcium buffer (140 mℳ NaCl, 5 mℳ KCl, 10 mℳ glucose, 10 mℳ HEPES, 2 mℳ CaCl2 and 1 mℳ MgCl2).
In all experiments concentrated stocks of peptides (IC3-C58, IC3-M28, IC3-C30, and IC3-C30) were made up in the appropriate buffer (with and without calcium), identical to that of the S100B protein.
were measured by loading with 5 μM Fura-2AM (Sigma-Aldrich) prepared in extracellular buffer with no calcium (125 mM NaCl, 5 mM KCl, 1.5 mM MgCl2, 20 mM HEPES, 10 mM glucose, pH7.4) for 30 min at 37 °C.
Third instar larvae were size-matched and dissected according to standard protocols in HL-3 buffer with low calcium levels (1.5 mM) on Sylgard plates (Verstreken et al., 2008).
To buffer intracellular calcium, we loaded cells with the calcium chelator BAPTA by incubating them with 20 μM BAPTA/AM for 30 min at 37°C.
420µL aliquots of truncated Lig proteins were loaded into the sample channels of double-sector, 12nm center pieces and 410µL of Tris buffer with 100 µM calcium chloride into the corresponding reference channels.
The vesicles were then diluted to 1 mg/ml final lipid concentration by adding 0.5 ml Tris buffer with calcium (NaCl 100 mM, Tris 10 mM, CaCl2 10 mM, pH 6.5), and the SiO2 surface was treated with oxygen plasma.
One mg/mL of LigAVar7'-8, LigAVar7'-10 LigAVar7'-12, LigBVar7'-81, LigBVar7'-10, LigAVar7'-13, LigBVar7'-8, LigBVar7'-9, LigBVar7'-10, LigBVar7'-11, or LigBVar7'-12 was dialyzed against prefiltered (0.22 µm Millipore filters) Tris buffer with 100 µM calcium chloride.
