Real-time Live-cell Flow Cytometry to Investigate Calcium Influx, Pore Formation, and Phagocytosis by P2X7 Receptors in Adult Neural Progenitor Cells

Abstract

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a method whereby live-cell flow cytometry is extended upon to analyze the multiple functions of P2X7 receptor activation in real-time. Using a time module installed on a flow cytometer, live-cell functionality can be assessed and plotted over a given time period to explore the kinetics of calcium influx, transmembrane pore formation, and phagocytosis. This simple method is advantageous as all three canonical functions of the P2X7 receptor can be assessed using one machine, and the gathered data plotted over time provides information on the entire live-cell population rather than single-cell recordings often obtained using technically challenging patch-clamp methods. Calcium influx experiments use a calcium indicator dye, while P2X7 pore formation assays rely on ethidium bromide being allowed to pass through the transmembrane pore formed upon high agonist concentrations. Yellow-green (YG) latex beads are utilized to measure phagocytosis. Specific agonists and antagonists are applied to investigate the effects of P2X7 receptor activity. Individually, these methods can be modified to provide quantitative data on any number of calcium channels and purinergic and scavenger receptors. Taken together, they highlight how the use of real-time live-cell flow cytometry is a rapid, cost-effective, reproducible, and quantifiable method to investigate P2X7 receptor function.

AP2RX7single nucleotide polymorphism haplotype promotes exon 7 and 8 skipping and disrupts receptor function

“…To determine whether EX7_EX8del protein isoforms form functional receptors, patch‐clamping and Ca + flux was used to measure channel function, and ethidium + uptake measured by time resolved flow cytometry was used to analyze pore function in HEK 293 cells transiently transfected with either P2X7A, P2X7L, or P2X7E. In addition, phagocytic function, in the absence of ATP, was analyzed by measuring the uptake of YG beads using flow cytometry . Channel and pore function were not detected in either P2X7E or P2X7L transfected cells (Figure A–C), however P2X7L showed the same level of phagocytic function compared to P2X7A (Figure D).…”

“…In addition, phagocytic function, in the absence of ATP, was analyzed by measuring the uptake of YG beads using flow cytometry. 8,9,39 Channel and pore function were not detected in either P2X7E or P2X7L transfected cells ( Figure 7A-C), however P2X7L showed the same level of phagocytic function compared to P2X7A ( Figure 7D). P2X7E, which is not trafficked to the cell surface, failed to confer phagocytic function ( Figure 7D).…”

Antinociceptive effect and anti-inflammatory activity of 1,4-naphthoquinones in mice

“…This work is a continuation of the study of the anti-inflammatory activity of the acyclic thioglucoside U-548 and its tetracyclic derivative U-286. In earlier research these compounds were found to exhibit antagonistic properties associated with blocking reaction pathways mediated by P 2 X 7 R in macrophage and neuronal cells in vitro [21,22]. Activation of P 2 X 7 R induces large-scale release of ATP into the extracellular space of tissues, due to its ability to form membrane macropores, as well as activate pannexin-1 hemichannels, thereby enhancing purinergic signaling and the inflammatory process [24].…”

“…Studied compounds U-286 [(2R,3R,4S,4aR,12aS)-2-hydroxymethyl-3,4-dihydroxy-3,4,4a,12a-tetrahydro-2H-naphtho(2, 3-b) pyrano(2,3-e)(1,4)-oxathiine-6, 11-dione] and U-548 [3-(β-D-glucopyranosyl-1-thio)-2methoxynaphthalene-1,4-dione] were synthesized in accordance with early described protocols [22]. Stocks of compounds were prepared in dimethyl sulfoxide (DMSO, Biolot, Russia) as 10 mmol/L solutions.…”

“…Compound U-286 was shown to inhibit the production of the mature form of IL-1β, as well as the proinflammatory cytokine tumor necrosis factor α (TNF-α) in RAW 264.7 macrophages. In addition, these compounds significantly reduced the activity of the proinflammatory enzyme cyclooxygenase 2 (COX-2) [22].…”