Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

Summary

Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-GloTM Luciferase Assay System from Promega.

Abstract

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane biogenesis occurs rapidly and in an inducible manner. We have found that phagocytosis of latex beads is practical for these purposes as cells rapidly synthesize membrane lipids to replenish membrane pools lost as wrapping material during particle engulfment. Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-Glo Luciferase Assay System from Promega.

Protocol

Cell Culture

1. Stock cultures of human embryonic kidney 293 (HEK293) cells are grown in 10-cm culture dishes in ‘medium A’, consisting of Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with a cocktail of antibiotics (100 units per ml of penicillin and 100 µg per ml of streptomycin sulfate) and 10% fetal bovine serum (FBS).
2. To set up cells for an experiment, the medium is sucked off, and the cells are washed two times with 5 ml of phosphate buffered saline (PBS) solution.
3. The PBS is then removed and replaced with 0.6 ml of a solution containing 0.25% trypsin.
4. The dish is incubated at 37˚C f....