B. burgdorferi cultures (GFP, wildtype or
Δp66) (20 μL/well) were plated in a 96 well
v-bottom plate (Corning). After the plate was centrifuged for 10 minutes at 1500 × g at 4°C and the supernatant was aspirated, the
B. burgdorferi were resuspended in FACS buffer (30 μL/well) (200 μL; 2% fetal bovine serum in PBS supplemented with
EDTA (1 mM, Thermo Fisher Scientific)) containing CV1-G4 or MIAP410 (10 μg/mL). Samples were incubated on ice (30 min) and protected from light. Upon incubation completion, the plate was washed twice with PBS (150 μL). For each wash, bacteria were pelleted prior to aspiration (1500 × g, 10 min, 4°C). After the second wash, the
B. burgdorferi were resuspended PBS (30 μL) containing
Alexa Fluor 647 anti-human IgG (1:200, Jackson ImmunoResearch) or
Alexa Fluor 647 anti-mouse IgG (1:200, Jackson ImmunoResearch). Samples were incubated on ice (30 min) and protected from light. Samples were again washed with PBS (2x) as described above.
B. burgdorferi were resuspended in 4% paraformaldehyde (100 μL, EMS) and incubated while protected from light (10 min, 25 °C). Samples were again washed with PBS (2x) as described above prior to resuspension in FACS buffer. Samples were protected from light until analysis on a
BD Fortessa.
Tal M.C., Hansen P.S., Ogasawara H.A., Feng Q., Volk R.F., Lee B., Casebeer S.E., Blacker G.S., Shoham M., Galloway S.D., Sapiro A.L., Hayes B., Torrez Dulgeroff L.B., Raveh T., Pothineni V.R., Potula H.H., Rajadas J., Bastounis E.E., Chou S., Robinson W.H., Coburn J., Weissman I.L, & Zaro B.W. (2024). P66 is a bacterial mimic of CD47 that binds the anti-phagocytic receptor SIRPα and facilitates macrophage evasion by Borrelia burgdorferi. bioRxiv.