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

Published: October 31, 2023

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…

Divulgazioni

The authors have nothing to disclose.

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

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Citazione di questo articolo
Quantifying Immune Cell Infiltration in Uropathogenic E. coli-Infected Mouse Bladder Tissue using Flow Cytometry. J. Vis. Exp. (Pending Publication), e21714, doi: (2023).

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