Lambda dg4 fluorescent excitation source

Erythrocyte calcium imaging experiments were performed as previously described15 (link). Briefly, blood was drawn and stored on ice in EGTA vacutainers. On the experimental day, we diluted whole blood 1:1,000 into NECM supplemented with 0.1% BSA and loaded with 5 μM Fluo-4 at 4 °C for at least an hour. Cells were then washed with buffer to remove excess dye and allowed to settle in a custom-build low-volume perfusion chamber, where a gravity-driven whole-chamber perfusion system was used to deliver Yoda1 and the calcium ionophore A23187 after erythrocytes loosely adhered to the uncoated glass-bottom chamber. For mechanical stimulation, we used long, tapered-tip (∼1.5–2 μm diameter) borosilicate pipettes (Sutter instruments, Novato, CA) forged with a Flaming/Brown P-97 puller (Sutter Instruments) and applied negative pressure to the cells using a High-speed Pressure Clamp device (ALA scientific, Farmingdale, NY). Imaging was performed using an Axio Observer (Zeiss) microscope fitted with a lambda DG4 fluorescent excitation source (Sutter Instruments). Images were recorded with an Orca Flash 4.0 camera (Hamamatsu Photonics) using MicroManager imaging software33 . Image analysis was performed with FIJI34 (link). HEK-P1KO cells were loaded with 2 μM Fura2-AM for 30 min at room temperature in NECM and imaged using the equipment described above.