An Assay to Determine Polymicrobial Biofilm Initiation on Virus-Infected Cells
Published: March 29, 2024
Abstract
Source: Plotkin, B. J. et al., Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi. J. Vis. Exp. (2016)
This video illustrates the initiation of polymicrobial biofilm formation on virus-infected cells by bacteria and fungi. Herpes simplex virus infection impedes bacterial attachment to the cells, preventing bacterial-fungal co-localization. In contrast, uninfected cells display bacterial-fungal co-localization and initiation of biofilm formation.
Protocol
1. HSV Strains and Handling NOTE: Recombinant non-spreading HSV-1(KOS) gL86 and HSV-2 (KOS) 333gJ- with beta-galactosidase reporter activity is used for the assay. Use virus from a single lot and store at -80 °C at a 1:1 ratio of Dulbecco's modified Eagle's medium (DMEM) with 20% fetal bovine serum (FBS) and skim milk until use. Before viral lot storage, determine virus concentration by o-nitrophenyl-β…
Representative Results
Figure 1. X-gal Staining Pictures of Dosage Dependent HSV-1 Infection in HeLa Cells. HeLa cells infected with HSV-1 at various MOI and X-Gal stained. (A) HeLa cells in well of 96 well plate with X-Gal stained mock-infected HeLa cell control; 20x initial magnification; (B) HeLa cells in well of 96 well plate with X-Gal stained HeLa cell infected with HSV-1 at…
Açıklamalar
The authors have nothing to disclose.
Materials
| C.albicans | |||
| BBL Sabouraud Dextrose | BD | 211584 | |
| Fungisel Agar | Dot Scientific | 7205A | |
| S.aureus | |||
| Mannitol Salt Agar | Troy Biologicals | 7143B | |
| Sheep blood agar | Troy Biologicals | 221239 | |
| Hela cells | |||
| 1xDMEM (Dubelcco's Modified Eagle Medium, with 4.5 g/L glucose and L-glutamine, without sodium pyruvate | Corning | 10-017-CM | |
| Gentamicin 50mg/ml | Sigma | 1397 | 50µg/ml final concentration in the complete DMEM |
| Trypsin EDTA (0.05% Trypsin, 0.53M EDTA)Solution 1X | Corning | 25-052-CI | |
| Fetal Bovine Serum | Atlanta Biologicals | S11150 | 10% final concentration in the complete DMEM |
| Other medium and reagents | |||
| ONPG | Thermo Scientific | 34055 | |
| Ultra-Pure X gal | Invitrogen | 15520-018 | |
| 1x HBSS (Hanks' Balanced Salt Solution) | Corning | 20-021-CV | |
| 1XPBS | Dot Scientific | 30042-500 | |
| RIPA Lysis | Life Technologies | 89901 | |
| Supplies | |||
| 96-Well plates | Evergreen Scientific | 222-8030-01F | |
| Culture tubes 100×13 | Thomas Scientific | 9187L61 |
Etiketler
Bu Makaleden Alıntı Yapın
An Assay to Determine Polymicrobial Biofilm Initiation on Virus-Infected Cells. J. Vis. Exp. (Pending Publication), e22008, doi: (2024).