Measuring endocytosis/phagocytosis of polystyrene or protein coated beads
- How do you measure how well macrophages kill bacteria (or whether your treatment alters killing)? This is a standard kill curve protocol and a workhorse of the Bowdish lab. Specifically written to measure S. pneumoniae killing, can be adapted for any bacteria and any type of macrophage (e.g. human, murine, alveolar, bone marrow,etc).
- Citation: Thevaranjan N, et al. Age-Associated Microbial Dysbiosis Promotes Intestinal Permeability, Systemic Inflammation, and Macrophage Dysfunction.Cell Host Microbe. 2017 Apr 12;21(4):455-466.e4. doi: 10.1016/j.chom.2017.03.002. PMID: 28407483
Preparation of mannosylated beads for phagolysosomal fusion assays
Measurement of binding to intact bacteria
- This protocol is used for “bacterial flow cytometry” (running bacteria through the flow cytometer rather than eukaroytic cells). We measure protein binding to the bacteria but it could easily be adapted to antibodies.
- Citation: Novakowski KE, et al.A naturally occurring transcript variant of MARCO reveals the SRCR domain is critical for function.Immunol Cell Biol. 2016 Aug;94(7):646-55. doi: 10.1038/icb.2016.20.
Class A Scavenger Receptor Blocking Protocol
- This protocol uses the scavenger receptor inhibitors dextran sulphate and poly I (and control ligands) to block scavenger receptor binding and is used whether ligand binding occurs by the class A scavenger receptors.
- Citation: Bowdish DM, et al. MARCO, TLR2, and CD14 are required for macrophage cytokine responses to mycobacterial trehalose dimycolate and Mycobacterium tuberculosis.PLoS Pathog. 2009 Jun;5(6):e1000474. doi: 10.1371/journal.ppat.1000474.