An Imaging Technique to Study the Calcium Response in Bacteria-Infected Cells

Abstract

Source: Smail, Y., et al. Imaging Ca²⁺ Responses During Shigella Infection of Epithelial Cells. J. Vis. Exp. (2018).

This video demonstrates a technique for detecting local and global calcium ion response upon Shigella infection of human epithelial cells. Shigella invades host cells and releases invasion effectors, impacting intracellular calcium levels. The amplitude and frequency of the local and global cytosolic calcium are studied using a fluorescent indicator.

Protocol

1. Preparations

1. Bacteria preparation
   1. Plate the bacteria—Shigella wild-type strain expressing the AfaE adhesin (M90T-AfaE)—onto a trypticase soy (TCS) agar plate containing 0.01% Congo red (CR) and incubate them for 18 h at 37 °C.
      Note: To increase their reproducibility, the Shigella plates obtained in step 1.1.1 are stored at 4 °C and should be used within a week because all colonies on CR medium will eventually turn red over time.
   2. Inoculate TCS broth pre-culture by picking 3 red colonies from the streak plates.
      Note:

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fluo-4 AM	Invitrogen	F14201
Metamorph version 7.7	Universal Imaging
CoolLED illumination system pE-2	Roper Scientific
Micro-dish 35 mm, high	IBIDI	81156
Trypticase Soy (TCS) broth	Thermofisher	B11768
TCS agar	Thermofisher	B11043
Congo red	Sigma-Aldrich	75768
M90T-AfaE	Sun et al. 2017		Shigella flexneri serotype V. expressing the AfaE adhesin
ipgD-AfaE	Sun et al. 2017		Isogenic ipgD mutant strain expressing the AfaE adhesin