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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.

<h2>Procedure</h2>
<h3>Labelling Bacteria</h3>
<h4>Preparation of Bacteria</h4>
# Grow bacteria as per usual, in order to produce sufficient bacteria for the phagocytosis assay, typically 10:1 to 200:1 bacteria:macrophage
# Transfer the desired number of bacteria to a 1.5 mL snap-cap tube and pellet using a 1 min/6,000 g centrifugation
# Remove the supernatant and suspend the bacteria in PBS
# 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
# Wash bacteria an additional 2 times in PBS, using 1 min/6,000 g centrifugations for each wash.

<h4>Surface Biotinylation</h4>
# Suspend the washed bacteria from above in 500 ul of PBS at a pH of 8.0 to 8.5
# Add 0.2 mg of NHS-biotin (e.g. EZ-Link NHS-LC-Biotin from Pierce)
# Incubate for 30 min at room temperature, rotating or with gentle agitation to keep bacteria suspended
# 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.
# Wash once with PBS, using 1 min/6,000 g centrifugations for each wash.

<h4>Fluorescent Labelling</h4>
# Suspend biotinylated bacteria from above step in 500 ul of PBS
# Add 0.5 ul of CellTrace Far-Red dye (Invitrogen) and mix (See Note 1)
# Incubate at room temperature for 20 min, protected from light, rotating or with gentle agitation to keep bacteria suspended
# 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.
# 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.

<hr>
<h3>Opsonization</h3>
In some experiments it may be necessary to opsonize the bacteria prior to use. This can be done with purified proteins or with serum.
# Prepare a working solution of the opsonin in an appropriate buffer:
## Serum: 20% in serum-Free DMEM, RPMI, HBSS or other calcium-containing media.
## Antibodies: most media will suffice, with or without calcium
## Recombinant proteins: match media to ion & other requirements of the protein
# 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.
# If required, wash bacteria in the same base media as the opsonin working solution was prepared in.

<h3>Phagocytosis Assays</h3>
''Protect the sample from light for all of these steps''
# Perform phagocytosis assay as per usual, for example, following our [[Synchronised Phagocytosis|Synchronised Phagocytosis]].
# At the end of the phagocytosis assay, fix the cells with 4% PFA in PBS, 20 min.
# Rinse 3 times with PBS, ideally adding a quenching agent (e.g. 100 mM glycine) to each wash.
# 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)
# Wash 3 times with PBS.
# Perform additional immunolabeling, counter-staining, etc as per the requirements of the experiment.
# 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)
<hr>
<h3>Notes</h3>
# 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.
# 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.
# This is a modified form of our apoptotic cell efferocytosis assays, published as: Evans AL, Blackburn JW, Yin C, Heit B. <ins>Quantitative Efferocytosis Assays</ins>. ''Methods Mol Biol''. 2017;1519:25-41. [http://www.ncbi.nlm.nih.gov/pubmed/27815871 [Pubmed]] [http://link.springer.com/protocol/10.1007%2F978-1-4939-6581-6_3 [Paper]]

Inside-out Labelling of Bacteria - Revision history

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