Quantifying Immune Cell Infiltration in Uropathogenic E. coli-Infected Mouse Bladder Tissue using Flow Cytometry

To quantify immune cell infiltration in uropathogenic E. coli-infected mouse bladder tissue, place the minced tissue in a digestion buffer containing collagenase and deoxyribonuclease.

Incubate with regular shaking to disrupt the tissue.

Collagenase digests the tissue's extracellular matrix, releasing cells. Deoxyribonuclease degrades interfering free DNA.

Post-incubation, add buffer with a chelating agent and fetal bovine serum to inactivate the digestive enzymes.

Pass the digested mixture through a strainer to remove tissue debris and connective tissue, obtaining a single-cell suspension.

Centrifuge and resuspend the cells in a buffer containing Fc-blocking antibodies. These antibodies bind to immune cell Fc receptors to prevent non-specific antibody binding.

Add fluorophore-tagged anti-CD45 antibodies, which bind to the transmembrane CD45 protein on immune cells.

Centrifuge and pass the resuspended cells through a cell strainer to remove cell aggregates.

Using a flow cytometer, measure the fluorescence signals from the fluorophore-conjugated antibodies on immune cells to quantify immune cell infiltration in the bladder.

To carry out flow cytometry on bladder tissue, after isolating the bladder as demonstrated, use scissors to mince the tissue well. Add 1 minced bladder per tube containing digestion buffer, then, shake the tube to wash the minced tissue in the digestion buffer, and keep it on ice until all the bladders are dissected and minced. Incubate the minced bladders at 37 degrees Celsius for 60 to 75 minutes with vigorous shaking by hand for 5 seconds every 15 minutes.

When the tissue has a glassy, transparent appearance resembling wet tissue paper, digestion is complete. Inactivate the digestion enzymes by adding 2 to 3 milliliters of ice-cold flow cytometry buffer, and gently mix. Then, place the tubes on ice.

To ensure a single-cell suspension and to remove any connective tissue, pass the contents of the tube through a 100-micrometer filter placed in a 15-milliliter conical tube. Then, with the end of a syringe plunger, gently press any remaining tissue through the filter. Wash the filter with an additional 2 milliliters of flow cytometry buffer, and keep the samples on ice. Then, wash the samples by centrifugation at 200 times g in 4 degrees Celsius for 7 minutes.

Resuspend the pellet in 100 microliters of flow cytometry buffer containing Fc block diluted to 5 micrograms per milliliter and transfer to a 96-well round-bottom plate. After 10 to 15 minutes, add 100 microliters of the desired antibody cocktail to each sample. Then, incubate the samples on ice, protected from light, for 30 to 45 minutes. Wash the samples by centrifugation at 200 times g in 4 degrees Celsius for 7 minutes.

Finally, resuspend the cell pellets in 200 microliters of flow cytometry buffer, and pass the samples through a 40-micrometer cell strainer on a 5-milliliter polystyrene tube just prior to acquisition on a flow cytometer.