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

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Flow Cytometric Analysis of Bladder Tissue

1. Prepare digestion buffer containing collagenase at 34 units/mL and DNase at 100 µg/mL, in PBS. Keep on ice. Prepare one 15 ml conical tube per animal to be analyzed and aliquot 1 ml digestion buffer per tube.
2. At predetermined time points, sacrifice mice using approved standard operating procedures (e.g., cervical dislocation or carbon dioxide overdose). Moisten the abdomen thoroughly with 70% ethanol to minimize contamination by fur.
3. Make an incision across the lower third of the mouse's abdomen of at least 2 cm using scissors and remove the entire bladder. Mince well with scissors, cutting the bladder into 2 - 3 mm² sized pieces, typically about 8 pieces.
   1. Add one minced bladder per tube containing digestion buffer. Shake to wash minced bladder tissue into the digestion buffer. Keep on ice until all bladders are dissected and minced.
4. Incubate minced bladders for 60 - 75 min at 37 °C, with vigorous shaking by hand for 5 s every 15 min. When the tissue has a glassy transparent appearance, resembling wet tissue paper, digestion is complete. Prolonged digestion will remove surface proteins from the cells needed for identification and should be avoided.
5. Inactivate the digestion enzymes by adding 2 - 3 mL of ice-cold flow cytometry buffer (PBS containing 2% fetal bovine serum (FBS) and 0.2 mM EDTA) and mix gently. Place tubes on ice.
6. To ensure a single-cell suspension and to remove any connective tissue, pass the contents of the tube through a 100 µm filter placed in a 15 mL conical tube. Gently press any remaining tissue through the filter with the end of a syringe plunger. Wash the filter with an additional 2 mL flow cytometry buffer. Keep samples on ice.
7. Wash samples by centrifugation for 7 min at 200 x g, 4 °C. Resuspend pellets in 100 µL flow cytometry buffer containing Fc block, diluted to 5 µg/mL, and transfer to a 96-well round bottom plate. After 10 - 15 min, add 100 µL desired antibody cocktail to each sample. Incubate samples for 30 - 45 min, on ice, protected from light.
   Note: Fc block is a reagent used to prevent the binding of the Fc portion of antibodies to cells expressing Fc receptors. Its purpose is to minimize nonspecific antibody binding. The determination of antibodies to use will be dependent upon the hypothesis being tested in the experiment and should be selected with input from the literature. In Figure 1, to measure the overall immune cell infiltration, an anti-CD45 antibody was employed, which is a pan-immune cell protein.
8. Wash samples by centrifugation for 7 min at 200 x g, 4 °C. Resuspend cell pellets in 200 µL flow cytometry buffer and pass samples through a 40 µm cell strainer on a 5 ml polystyrene tube just prior to acquisition on a flow cytometer.
9. Collect the entire sample on the flow cytometer. A good digestion will yield 200,000 - 400,000 cells, with 8,000 - 10,000 CD45⁺ immune cells from naïve mouse bladders, while infected organs will contain more cells (Figure 1).
   Note: Samples prepared for flow cytometric analysis cannot be used to evaluate CFU. It is not recommended to split the bladder into two parts as no evidence exists to demonstrate that uropathogenic E. coli (UPEC) colonization or immune cell infiltration is uniform throughout the tissue.

Results

Figure 1: Immune Cell Infiltration is Increased in Female Mice in Response to UTI. Six- to eight-week-old female and male C57Bl/6 mice were instilled or not with 10⁷ CFU of UPEC. Representative dot plots depict total bladder cells with CD45⁺ immune cells gated in pink from naïve or infected mice. The graph shows the absolute number of CD45⁺ i...

Materials

Name	Company	Catalog Number	Comments
Syringes	B Braun	9166017V
Thumb (Adson) forceps	Fine Science Tools	11006-12
5 mL tubes, polypropylene	Falcon	352063
Tissue Ruptor disposable probes	Qiagen	990890
15 mL flip cap tubes	Thermo Scientific	362694
DNAse	Invitrogen	18047019
100 µM MACS SmartStrainers	Miltenyi	130-098-463	Compatible with 15 mL tubes, preferred
100 µm cell strainers	Falcon	352360	Compatible with 50 mL tubes, if MACS Smart Strainers are not available
96 well plate	Falcon	353077
Fc block	BD Pharmingen	553142	Anti-CD16/CD32
Anti-mouse CD45 antibody	BD Biosciences	561487	Many different fluorescent conjugation options are available
5 mL tubes polystyrene with filter cap	Falcon	352235
Hand held homogenizer	Qiagen	9001272	TissueRuptor
Flow cytometer	BD Biosciences	N/A	Fortessa SORP
Flow cytometry software	BD Biosciences	N/A	Diva