Rabbit anti hamster igg

Naïve CD4⁺ T cells as well as T_H1 and T_H2 cells from WT and Orai1^fl/flCd4Cre mice were loaded with 1 μM Fura-2-AM (Molecular Probes, F1221) for 30 min at room temperature, washed, and attached to 96-well plates (BD Falcon, 353219) coated with 0.01% (v/v) poly-l-Lysine (Sigma-Aldrich, P8920). At the beginning of each experiment, cells were kept in Ca²⁺-free Ringer solution [155 mM NaCl, 4.5 mM KCl, 3 mM MgCl₂, 10 mM d-glucose, and 5 mM Na-Hepes (pH 7.4)], followed by treatment with 1 μM thapsigargin (Sigma-Aldrich, 586005) to deplete ER Ca²⁺ stores. Cells were subsequently perfused with Ca²⁺-containing Ringer solution [155 mM NaCl, 4.5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM d-glucose, and 5 mM Na-Hepes (pH 7.4)] at the indicated time points to induce SOCE. Alternatively, cells were stimulated with anti-CD3 (1 μg/ml; clone 2C11) for 30 min during the loading with Fura-2-AM. Cells were then washed and kept in Ca²⁺-free Ringer solution at the beginning of the recording, followed by CD3 cross-linking with rabbit anti-hamster IgG (1 μg/ml; MP Biomedicals, MP0855398), and addition of Ca²⁺-containing Ringer solution. Fura-2 fluorescence was measured at an emission wavelength of 510 nm after excitation at 340 nm and 380 nm using FlexStation 3 (Molecular Devices) and plotted as baseline-normalized F340/F380 emission ratio.