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

Abstract

Source: Zychlinsky Scharff, A. et al., Urinary Tract Infection in a Small Animal Model: Transurethral Catheterization of Male and Female Mice. J. Vis. Exp. (2017)

In this video, we demonstrate a flow-cytometry-based technique to quantify CD45⁺ immune cells in Escherichia coli-infected mouse bladder to measure immune cell infiltration.

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 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. At predetermined time points, sacrifi…

Representative 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

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