Pluronic f27

Ca²⁺ measurements were performed essentially as described previously [20 (link), 78 (link), 79 (link)]. SH-SY5Y cells were loaded with 2 μM Fluo4-AM and/or Rhod2-AM dye (Invitrogen) in external solution (145 mM NaCl, 2 mM KCl, 5 mM NaHCO₃, 1 mM MgCl₂, 2.5 mM CaCl₂, 10 mM glucose, 10 mM Na-HEPES, pH 7.25) containing 0.02% Pluronic-F27 (Invitrogen) for 15 min at 37 °C, followed by washing in external solution for 15 min at 37 °C. Fluo4 and Rhod2 fluorescence were timelapse recorded (1 s intervals) at 37 °C using either an Axiovert S100 microscope (Zeiss) driven by MetaMorph (Molecular Dynamics) and equipped with GFP (Fluo4) and DsRed (Rhod2) filtersets (Chroma Technology), a 40× Plan-Neofluar 1.3NA objective (Zeiss), and a Photometrics Cascade-II 512B36 EMCCD camera or a Nikon Ti-E microscope using a CFI Plan Apo VC 20× objective and Nikon Andor Neo sCMOD high-resolution camera and appropriate filter sets. The cells were kept under constant perfusion with external solution (0.5 ml/min). Inositol 1,4,5-trisphosphate (IP3) receptor-mediated Ca²⁺ release from ER stores was triggered by application of 100 μM Oxotremorine-M for 2 min. Ca²⁺ levels were calculated as relative Rhod2 or Fluo4 fluorescence compared to baseline fluorescence (F/F₀) at the start of the measurement. Oxotremorine-M was from Santa Cruz Biotechnology and was dissolved in water.

Measurement of Ca²⁺ mobilization was previously described (45 (link)). Briefly, a total of 1 x10⁶ ER^T2-SLP65, pre-BCR positive TKO cells were loaded with Indo-1 AM (Invitrogen) using Pluronic F27 (Invitrogen). ER^T2-SLP65 function was induced by the addition of 2 µM 4-OHT or the respective amount of its solvent Ethanol (Et) as control. Pre-BCR expression and Ca2+ mobilization was acquired at a FACS LSR Fortessa flow cytometer (BD).