Bacterial Cell Staining and Preparation for Immunological Studies
Published: November 30, 2023
Abstract
Source: Yang, J. et al., Automated, High-Throughput Detection of Bacterial Adherence to Host Cells. J. Vis. Exp. (2021)
This video demonstrates the staining and preparation of bacterial cells for immunological studies using red fluorescent dye. The stained bacteria are diluted and resuspended in a growth medium to achieve the desired bacterial concentration for host cell infection, representing the multiplicity of infection.
Protocol
1. Bacterial growth and staining Perform all bacterial work in a Biosafety Cabinet, Biosafety Level 2 laboratory. Inoculate all bacterial cultures, including Pseudomonas aeruginosa (PAO1), Escherichia coli, Listeria monocytogenes, Bacillus subtilis, etc., from the frozen glycerol stock and grow them in Tryptic Soy Broth (TSB, 3 mL) in a shaking incubator at 37 °C overnight maintained at 250 rpm. T…
Disclosures
The authors have nothing to disclose.
Materials
| 10x PBS | VWR | 45001-130 | |
| BactoView Live Red | Biotium | 40101 | Bacteria staning dye |
| Centrifuge | Eppendorf | 5810R | |
| E. coli | Laboratory stock | ||
| F-12k medium | ATCC | 302004 | A549 cell culture medium |
| Fetal bovine serum | Corning | 35-016-CV | |
| L. monocytogenes | NIST collections | ||
| OD600 DiluPhotometer | IMPLEN | ||
| P. aeruginosa | Dr. Lori Burrows laboratory stock | ||
| P. aeruginosa ΔpilA | Dr. Lori Burrows laboratory stock | ||
| S. agalactiae | NIST collections | ||
| S. aureus | BEI | NR-46543 | |
| S. aureus ΔsaeR | BEI | NR-48164 | |
| S. rubidaea | NIST collections | ||
| Typical soy broth | Growcells | MBPE-4040 |
Cite This Article
Bacterial Cell Staining and Preparation for Immunological Studies. J. Vis. Exp. (Pending Publication), e21805, doi: (2023).