An Assay to Study the Impact of Extracellular Matrix Stiffness on Bacterial Infection

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…

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/