An Imaging Technique to Study the Calcium Response in Bacteria-Infected Cells
Published: October 31, 2023
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 Bacteria preparation 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 wil…
Offenlegungen
The authors have nothing to disclose.
Materials
| 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 |
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An Imaging Technique to Study the Calcium Response in Bacteria-Infected Cells. J. Vis. Exp. (Pending Publication), e21664, doi: (2023).