An In Vitro Assay to Study the Interaction between Neutrophils and Biofilm

Abstract

Source: Rana, P. S. J. B., et al. Standardized In vitro Assays to Visualize and Quantify Interactions between Human Neutrophils and Staphylococcus aureus Biofilms. J. Vis. Exp. (2022)

This video demonstrates an in vitro assay to study neutrophil-biofilm interaction. The pathogenic bacteria embedded in the biofilm and isolated neutrophils were tagged using fluorescent labels, and the immune response by the neutrophils and the evasion of the immune response by the bacteria were assessed using a fluorescence microscope.

Protocol

1. Preparation of in vitro biofilm Obtain isolated colonies of S. aureus from a cryopreserved stock using a streak-plate technique on a nutrient-rich agar plate, such as Tryptic Soy Agar (see Table of Materials). Coat individual wells of a 96-well plate with 100 µL of 0.001% (v/v) poly-L-Lysine (PLL) diluted in sterile H2O and incubate at room temperature for 30 min. Aseptically, aspirate the PLL solution using a vacuum-assisted asp…

Materials

0.9% sodium chloride irrigation, USP	Baxter	2F7124	Endotoxin-free; Used for isolation of neutrophils
150 mL rapid-flow filter unit	Thermo Scientific	565-0020
200 proof ethanol	VWR	89125-188
3 mL syringe	BD	309657	Used for blood draw
50 mL conical centrifuge tubes	Thermo Scientific	339652
60 mL syringe	BD	309653	Used for blood draw
Agar	Fisher Bioreagents	BP1423-2
Alcohol swab	BD		Used for blood draw
Band-aids			Used for blood draw
BD Bacto Tryptic Soy Broth	BD	DF0370-07-5	Combine with 1.5% agar to make Tryptic Soy Agar
Cell counter	Bal Saupply	202C
CellTracker blue CMCH	Invitrogen	C2111	Blue CMAC Dye (BCD)
Clear bottom 96-well flat bottom polystyrene plates	Costar	3370
Cotton gauze	Fisherbrand	13-761-52	Used for blood draw
Culture tubes	Fisherbrand	14-961-27	Borosilicate Glass 13 x 100 mm
D-(+)-glucose	Sigma	G-8270
Dextran from Leuconostoc spp.	Sigma	31392-250G	Used for isolation of neutrophils
Dulbecco's phosphate-buffered saline (DPBS) 1x	Gibco	14190-144
Ethidium homodimer-1	Invitrogen	L3224 B
Ficoll-Paque plus	Cytiva	17144003	Used for isolation of neutrophils (density gradient medium)
Hanks' balanced salt solution (HBSS) 1x	Corning cellgro	21-022-CV	Without calcium, magnesium, and phenol red
Hemocytometer	Bright Line
Heparin	Novaplus	NDC 63323-540-57	1000 USP units/mL, Used for blood draw
IMARIS 9.8	Oxford Instruments		Microscopy image analysis software
Minimal essential media (MEM) Alpha 1x	Gibco	41061-029
Needle (23 G1)	BD	305145	Used for blood draw
Nikon Eclipse Ti2	Nikon
NIS-Elements	Nikon		Quantification of dead neutrophils
Poly-L-lysine solution	Sigma	P4707-50ML
Sodium chloride	Fisher Bioreagents	BP358-10	Used for neutrophil isolation
Sterile water for irrigation, USP	Baxter	2F7114	Endotoxin-free; Used for neutrophil isolation
Surflo winged infusion set	Terumo	SC*19BLK	19 G x 3/4", used for blood draw
Trypan blue stain (0.4%)	Gibco	15250-061
Turnicate			Used for blood draw
UltraPure distilled water	Invitrogen	10977015
White opaque 96-well plates	Falcon	353296	Tissue culture treated and flat bottom plate
μ-Slide VI 0.4	Ibidi	80601	μ-channel slide