Goat anti armenian hamster igg antibodies

Jurkat cells and LAT mutant variants were washed with PBS twice, and loaded with 1 μM Indo-1 AM calcium indicator dye (Thermo Fisher Scientific #I1223) at 37 °C for 30 min in RPMI medium. After loading with Indo-I, cells were washed with PBS twice, and transferred into 96 well plates. Changes in the fluorescence ratio (violet/blue) following the addition of anti-CD3 (OKT3 clone) or ionomycin were recorded using a Flex Station II (Molecular Probes) using SoftMax Pro software. Data were imported into GraphPad Prism software for analysis and production of graphs. Calcium responses in primary thymocytes from bone marrow chimera studies were analyzed by flow cytometry. Cells were loaded with 1.5 μM Indo-I AM at 37 °C in RPMI medium supplemented with 5% fetal bovine serum for 30 min. After loading with Indo-I, cells were stained with CD4, CD8, and CD45.2 antibodies. Cells were analyzed by flow cytometry and stimulated with 1 μg/ml anti-CD3 (clone 145-2C11), followed by crosslinking with 50 ng/ml goat anti-Armenian hamster IgG antibodies (Jackson ImmunoResearch #127-005-099). Change in intracellular calcium concentration were monitored as the ratio of Indo-I (blue/violet) and displayed as a function of time.