In Vitro Evaluation of Bacterial Killing by Neonatal Mouse Splenic Phagocytes

Samenvatting

Source: Seman, B. G., et.al. Neonatal Imaging Model of Gram-Negative Bacterial Sepsis. J. Vis. Exp. (2020)

This video demonstrates the isolation of neutrophils and inflammatory monocytes from neonatal mouse spleens and their application in a luminescent bacterial killing assay. It covers mechanical dissociation, red blood cell lysis, magnetic bead–based cell enrichment, bacterial incubation, and antibiotic protection. Timed luminescence readings indicate initial bacterial internalization followed by immune-mediated clearance.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. In vitro bacterial killing assay

1. Remove the spleen from an uninfected neonatal pup and place it in a 40 µm nylon basket within a sterile 60 mm Petri dish. Repeat this and pool spleens into one tube to be harvested and homogenized together.
2. Add 5 mL of phosphate-buffered saline (PBS) supplemented with 10% fetal bovine serum (FBS).
3. Disaggregate the tissue using a sterile 3 mL syringe plunger until a single cell suspension is created.
4. Collect the single-cell suspension outside of the nylon basket, transfer to a 15 mL centrifuge tube, and pellet cells at 350 x g for 5 min.
5. Suspend the cells in a red blood cell lysis buffer (2 mL for up to 7-8 spleens) and let it stand for 5 min at room temperature to eliminate erythrocytes.
6. Wash splenocytes with PBS and pellets as above.
7. Suspend the splenocytes in 0.25 mL of PBS supplemented with 0.5% BSA and 2 mM EDTA according to expected cell yield.
8. Count the splenocytes using a hemocytometer or other appropriate application.
9. Isolate Ly6B.2⁺ (myeloid population of granulocytes/inflammatory monocytes) cells with immunomagnetic beads.
10. Seed Ly6B.2⁺ cells at a density of 1 x 10⁵ cells per well in a black or white 96-well plate in a volume of 0.1 mL of DMEM that contains 10% FBS, 2 mM glutamine, and 25 mM HEPES (complete medium).
11. Enumerate bioluminescent E. coli and prepare the bacterial inoculum at the desired multiplicity of infection (MOI) in a final volume of 0.1 mL. This is best done by making what is necessary for all wells at a common MOI in batch.
12. Add 0.1 mL of bacterial inoculum or complete medium alone as a control. Incubate the multi-well plate at 37 °C and 5% CO₂ for 1 h.
13. Replace the media with 0.2 mL of fresh complete media that contains gentamicin (100 µg/mL) by gently removing media with a pipette and adding fresh media with a new pipette tip. Return the culture to incubation for an additional 2 h.
14. At 3 h post-infection, measure the luminescence in each well of the lidded culture plate from the bottom using a plate reader and then return the culture to incubation.
15. Repeat measurements of luminescence at other desired time points.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
ACK Lysis Buffer	Gibco	LSA1049201	Bacterial clearance assay
Anti-Ly-6B.2 Microbeads	Miltenyi Biotec	130-100-781	Cell isolation
Escherichia coli O1:K1:H7-lux (expresses luciferase)	N/A	N/A	Constructed in-house at WVU
Isolation Buffer	Miltenyi Biotec	N/A	Bacterial clearance assay
LB Broth, Lennox	Fisher BioReagents	BP1427-500	Bacterial growth
EASYstrainer (Nylon Basket)	Greiner Bio-one	542 040	Cell strainer
SpectraMax iD3	Molecular Devices	N/A	Plate reader