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.
1. Manufacturing Thin Two-layered Polyacrylamide (PA) Hydrogels on Multi-Well Plates
2. Human Microvascular Endothelial Cell Culture and Seeding on Hydrogels
3. Infection of Human Microvascular Endothelial Cells with L. monocytogenes
4. Flow Cytometry to Quantify Extracellular-matrix-stiffness Dependent Susceptibility of Host Cells to Infection
Table 1. Composition of polyacrylamide (PA) hydrogels of varying stiffness. In this table, the percentage of stock 40% acrylamide solution and the percentage of stock 2% bis-acrylamide solution to achieve a given stiffness (Young's modulus, E) are indicated in different columns.
Young's modulus (E, kPa) | Acrylamide % (from 40% stock) | Bisacrylamide % (from 2% stock) |
0.6 | 3 | 0.045 |
3 | 5 | 0.075 |
10 | 10 | 0.075 |
20 | 8 | 0.195 |
70 | 10 | 0.45 |
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 glass coverslip to enable polymerization. D. The coverslip is removed with a needle syringe. E. 2.4 µL of a PA solution with microbeads is added on top of the bottom layer and capped with a circular glass coverslip. F. A buffer is added in the well and the coverslip is removed. G. UV irradiation for 1 h ensures sterilization. H. A Sulfo-SANPAH-containing solution is added on the gels, which are then placed under UV for 10 min. I. The hydrogels are washed with a buffer and then incubated overnight with collagen I. J. The hydrogel is equilibrated with cell media. K. The host cells are seeded. L. Lm bacteria are added to the solution and the infection is synchronized via centrifugation. M. 1 h post-infection bacteria in the solution are washed away and media supplemented with an antibiotic is added. N. At 4 h post-infection, Lm (JAT985) starts fluorescing. O. HMEC-1 cells are detached from their matrix and the solutions are transferred to tubes to perform flow cytometry measurements. Note that days and approximate times for each step of the assay are also indicated. This figure has been modified from Bastounis and Theriot.
Figure 2: AFM measurements of PA hydrogel stiffness and beads' distribution. A. Data show the expected Young's modulus (measure of stiffness) of the PA hydrogels, given the amount of acrylamide and bis-acrylamide used versus the Young's modulus measured through AFM (N = 5 – 6). The horizontal bars depict the mean. The stiffness of the 0.6 kPa hydrogels could not be measured because the hydrogels were very soft and adhered to the AFM tip. B. This is a phase image of confluent HMEC-1 cells and the corresponding image of the beads embedded on the uppermost surface of a soft 3 kPa-PA hydrogel. The HMEC-1 were seeded for 24 h at a concentration of 4 x 105 cells per well. C. This image is the same as Figure 2B but for cells residing on a stiff 70-kPa PA hydrogel.
The authors have nothing to disclose.
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/ |