Protocol for isolating and culturing human monocytes from peripheral blood and measuring intracellular calcium...

Here, we present a protocol for isolating and culturing human monocytes from peripheral blood and measuring intracellular calcium ([Ca²⁺]ᵢ) dynamics using the fluorescent indicator Fura-2 AM. We describe steps for separating peripheral blood mononuclear cells (PBMCs) by density gradient centrifugation and purifying monocytes using magnetic-activated cell sorting (MACS). We then detail procedures for culturing purified monocytes and subjecting them to real-time Ca²⁺ imaging to evaluate purinergic receptor activation, particularly P2X7 receptors, and downstream Ca²⁺ signaling in primary human monocytes. For complete details on the use and execution of this protocol, please refer to Garrosa-Jiménez et al.¹.