An Assay to Study the Impact of Extracellular Matrix Stiffness on Bacterial Infection
Published: March 29, 2024
Abstract
Source: Bastounis, E. E., et al. A Multi-well Format Polyacrylamide-based Assay for Studying the Effect of Extracellular Matrix Stiffness on the Bacterial Infection of Adherent Cells. J. Vis. Exp. (2018).
This video demonstrates an assay to study the effect of extracellular matrix (ECM) stiffness on bacterial infection of adherent cells. Polyacrylamide hydrogels of varying stiffness, overlaid with collagen, are layered in a multi-well plate to mimic the stiffness of physiological ECM. Upon overlaying human endothelial cells on the hydrogels and infecting the cells with Listeria monocytogenes bacteria engineered to express a fluorescent protein once intracellular, flow cytometry is used to compare internalized bacteria in cells residing on stiffer hydrogels versus softer hydrogels.
Protocol
1. Manufacturing Thin Two-layered Polyacrylamide (PA) Hydrogels on Multi-Well Plates Polyacrylamide hydrogel fabrication NOTE: See Figure 1. Prepare aqueous solutions that contain 3 – 10% of 40% stock acrylamide solution (see Table of Materials) and 0.06 – 0.6% of 2% stock bis-acrylamide solution (see Table of Materials) to manufacture hydrogels of tunable stiffness ranging f…
Representative Results
Figure 1: Bacterial infection assay of host cells residing on thin two-layered fluorescent bead-embedded polyacrylamide (PA) hydrogels of varying stiffness. A. Glass coverslips are chemically modified to enable hydrogel attachment. B. 3.6 µL of PA mixtures are deposited on the glass bottoms. C. The mixture is covered with a 12-mm circular glas…
Offenlegungen
The authors have nothing to disclose.
Materials
| Reagents | |||
| Sodium hydroxide pellets | Fisher | S318-500 | |
| (3-Aminopropyl)triethoxysilane | Sigma | A3648 | |
| 25% gluteraldehyde | Sigma | G6257-100ML | |
| 40% Acrylamide | Sigma | A4058-100ML | |
| Bis-acrylamide solution (2%w/v) | Fisher Scientific | BP1404-250 | |
| Fluorospheres carboxylate-modified microspheres, 0.1 μm, yellow-green fluorescent (505/515) | Invitrogen | F8803 | |
| Ammonium Persulfate | Fisher | BP17925 | |
| TEMED | Sigma | T9281-25ML | |
| Sulfo-SANPAH | Proteochem | c1111-100mg | |
| Collagen, Type I Solution from rat | Sigma | C3867-1VL | |
| Dimethyl sulfoxide (DMSO) | J.T. Baker | 9224-01 | |
| HEPES, Free acid | J.T. Baker | 4018-04 | |
| Leibovitz's L-15 medium, no phenol red | Thermofischer | 21083027 | |
| MCDB 131 Medium, no glutamine | Life technologies | 10372019 | |
| Foundation Fetal Bovine Serum, Lot: A37C48A | Gemini Bio-Prod | 900108 500ml | |
| Epidermal Growth Factor, EGF | Sigma | E9644 | |
| Hydrocortisone | Sigma | H0888 | |
| L-Glutamine 200mM | Fisher | SH3003401 | |
| DPBS 1X | Fisher | SH30028FS | |
| Gentamicin sulfate | MP biomedicals | 194530 | |
| Cloramphenicol | Sigma | C0378-5G | |
| DifcoTM Agar, Granulated | BD | 214530 | |
| BBL TM Brain-heart infusion | BD | 211059 | |
| Hoechst 33342, trihydrochloride, trihydrate – 10 mg/ul solution in water | Invitrogen | H3570 | |
| Formaldehyde 16% EM grade | Electron microscopy | 15710-S | |
| Anti-Listeria monocytogenes antibody | Abcam | ab35132 | |
| Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor® 546 conjugate | Thermofischer | A-11035 | |
| 0.25% trypsin-EDTA , phenol red | Thermofischer | 25200056 | |
| COLLAGENASE FROM CLOSTRIDIUM HISTOLYTIC | Sigma | C8051 | |
| Streptomycin sulfate | Fisher Scientific | 3810-74-0 | |
| Sucrose | Calbiochem | 8510 | |
| Sodium dodecyl sulfate | Thermofischer | 28364 | |
| MES powder | Sigma | M3885 | |
| KCl | J.T. Baker | 3040-05 | |
| MgCl2 | J.T. Baker | 2444-1 | |
| EGTA | Acros | 40991 | |
| Disposable lab equipment | |||
| 12 mm circular glass coverslips | Fisherbrand | 12-545-81 | No. 1.5 Coverslip | 10 mm Glass Diameter | Uncoated |
| Glass bottom 24 well plates | Mattek | P24G-1.5-13-F | |
| 5 ml polystyrene tubes with a 35 μm cell strainer cap | Falcon | 352235 | |
| T-25 flasks | Falcon | 353118 | |
| 50 ml conical tubes | Falcon | 352070 | |
| 15 ml conicals tubes | Falcon | 352196 | |
| Disposable Serological Pipettes (1 ml, 2 ml, 5 ml, 10 ml, 25 ml) | Falcon | 357551 | |
| Pasteur Glass Pipettes | VWR | 14672-380 | |
| Pipette Tips (1-200 μl, 101-1000 μl) | Denville | P1122, P1126 | |
| Powder Free Examination Gloves | Microflex | XC-310 | |
| Cuvettes bacteria | Sarstedt | 67.746 | |
| Razors | VWR | 55411-050 | |
| Syringe needle | BD | 305167 | |
| 0.2um sterilizng bottles | Thermo Scientific | 566-0020 | |
| 20 ml syringes | BD | 302830 | |
| 0.2um filters | Thermo Scientific | 723-2520 | |
| wooden sticks | Grainger | 42181501 | |
| Saran wrap | Santa Cruz Biotechnologies | sc-3687 | |
| Plates bacteria | Falcon | 351029 | |
| Large/non-disposable lab equipment | |||
| Tissue Culture Hood | Baker | SG504 | |
| Hemacytometer | Sigma | Z359629 | |
| Bacteria incubator | Thermo Scientific | IGS180 | |
| Tissue culture Incubator | NuAire | NU-8700 | |
| Vacuum chamber/degasser | Belart | 42025 | 37 °C and 5% CO2 |
| Inverted Nikon Diaphot 200 epifluorescence microscope | Nikon | NIKON-DIAPHOT-200 | |
| Cage Incubator | Haison | Custom | |
| Scanford FACScan analyzer | Stanford and Cytek upgraded FACScan | Custom | |
| Pipette Aid | Drummond | 4-000-110 | |
| Pipettors (10 μl, 200 μl, 1000ul) | Gilson | F144802, F123601, F123602 | |
| pH meter | Mettler Toledo | 30019028 | |
| forceps | FST | 11000-12 | |
| 1 L flask | Fisherbrand | FB5011000 | |
| Autoclave machine | Amsco | 3021 | |
| Stir magnet plate | Bellco | 7760-06000 | |
| Magnet stirring bars | Bellco | 1975-00100 | |
| Spectrophotometer | Beckman | DU 640 | |
| Scanford FACScan analyzer | Cytek Biosciences | Custom Stanford and Cytek upgraded FACScan | |
| Software | |||
| Microscope Software (μManager) | Open Imaging | ||
| Matlab | Matlab Inc | ||
| Flowjo | FlowJo, LLC | ||
| Automated image analysis software, CellC | https://sites.google.com/site/cellcsoftware/ | The software is freely available. Eexecutable files and MATLAB source codes can be obtained at https://sites.google.com/site/cellcsoftware/ |
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Diesen Artikel zitieren
An Assay to Study the Impact of Extracellular Matrix Stiffness on Bacterial Infection. J. Vis. Exp. (Pending Publication), e22005, doi: (2024).