Inside-out Labelling of Bacteria

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Many phagocytosis assays require that bacteria be labelled such that internalised bacteria can be clearly delineated from those merely bound to the cell surface. This is achieved through dual-labelling, using a fluorescent probe to label all bacteria in the sample, and using surface biotinylation and streptavadin to label non-internalised bacteria.

Procedure

Labelling Bacteria

Preparation of Bacteria

  1. Grow bacteria as per usual, in order to produce sufficient bacteria for the phagocytosis assay, typically 10:1 to 200:1 bacteria:macrophage
  2. Transfer the desired number of bacteria to a 1.5 mL snap-cap tube and pellet using a 1 min/6,000 g centrifugation
  3. Remove the supernatant and suspend the bacteria in PBS
  4. If required, kill the bacteria. Methods for this can vary, but incubation at 70C for 10 min, followed by room temperature incubation with 2% PFA for 10 min, works for most bacterial species
  5. Wash bacteria an additional 2 times in PBS, using 1 min/6,000 g centrifugations for each wash.

Surface Biotinylation

  1. Suspend the washed bacteria from above in 500 ul of PBS at a pH of 8.0 to 8.5
  2. Add 0.2 mg of NHS-biotin (e.g. EZ-Link NHS-LC-Biotin from Pierce)
  3. Incubate for 30 min at room temperature, rotating or with gentle agitation to keep bacteria suspended
  4. Quench any unreacted biotin with the addition 500 ul of an amine-containing buffer (e.g. PBS + 100 mM glycine, LB media, etc). Incubate for 10 min at room temperature with rotation or gentle agitation.
  5. Wash once with PBS, using 1 min/6,000 g centrifugations for each wash.

Fluorescent Labelling

  1. Suspend biotinylated bacteria from above step in 500 ul of PBS
  2. Add 0.5 ul of CellTrace Far-Red dye (Invitrogen) and mix (See Note 1)
  3. Incubate at room temperature for 20 min, protected from light, rotating or with gentle agitation to keep bacteria suspended
  4. Quench any unreacted CellTrace with the addition 500 ul of an amine-containing buffer (e.g. PBS + 100 mM glycine, LB media, etc). Incubate for 10 min at room temperature with rotation or gentle agitation.
  5. Wash twice with PBS, using 1 min/6,000 g centrifugations for each wash, then suspend at the desired concentration.
Note: Labelled killed cells can be kept refrigerated for up to 5 days; live cells should be used immediately after preparation.

Opsonization

In some experiments it may be necessary to opsonize the bacteria prior to use. This can be done with purified proteins or with serum.

  1. Prepare a working solution of the opsonin in an appropriate buffer:
    1. Serum: 20% in serum-Free DMEM, RPMI, HBSS or other calcium-containing media.
    2. Antibodies: most media will suffice, with or without calcium
    3. Recombinant proteins: match media to ion & other requirements of the protein
  2. Suspend the labelled bacteria in the opsonization solution, incubate for 20 min at either room temperature or 37C. Rotate/agitate the tube frequently to keep bacteria in suspension.
  3. If required, wash bacteria in the same base media as the opsonin working solution was prepared in.

Phagocytosis Assays

Protect the sample from light for all of these steps

  1. Perform phagocytosis assay as per usual, for example, following our Synchronised Phagocytosis.
  2. At the end of the phagocytosis assay, fix the cells with 4% PFA in PBS, 20 min.
  3. Rinse 3 times with PBS, ideally adding a quenching agent (e.g. 100 mM glycine) to each wash.
  4. Add fluorescently-labelled streptavadin in PBS (1:1000 dilution of a 1 mg/ml stock), incubate for 5 min at room temperature. (See Note 1)
  5. Wash 3 times with PBS.
  6. Perform additional immunolabeling, counter-staining, etc as per the requirements of the experiment.
  7. Mount on a slide using an anti-fade mounting media and image using the appropriate DIC/phase contrast and fluorescence channels for the labels used. (See Note 2)

Notes

  1. We recommend using CellTrace Far-Red and a blue-labelled streptavadin (e.g. pacific blue, alexa-405, dylight-405) for these experiments, as this leaves the GFP and RFP channels of the microscope free for fluorescent transgenes or conventional immunostaining/counter-staining. Both other combinations of streptavadin and CellTrace colours can be used.
  2. Internalised (phagocytosed) bacteria will be labelled with only the CellTrace dye, why extracellular bacteria will be dual-labelled with CellTrace and Streptavadin. Biotin-positive bacteria in-contact with a cell (determined by DIC or another fluorescent marker) are considered bound.
  3. This is a modified form of our apoptotic cell efferocytosis assays, published as: Evans AL, Blackburn JW, Yin C, Heit B. Quantitative Efferocytosis Assays. Methods Mol Biol. 2017;1519:25-41. [Pubmed] [Paper]
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