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.
1. Preparation of in vitro biofilm
2. Neutrophil isolation
NOTE: Neutrophils were isolated following a previously published method with minor changes. This isolation protocol combines density gradient centrifugation first, followed by 3% dextran sedimentation. This section only covers the overall neutrophil isolation protocol, focusing on the changes made to the published protocol. Furthermore, the protocol outlined below is one of the many methods that can isolate neutrophils and can be substituted as needed. Other methods for isolating neutrophils include the use of cell separation media or magnetic antibody cell separation.
3. Imaging biofilm-neutrophil interactions
The authors have nothing to disclose.
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 |