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…
Divulgations
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