Protocol for the rapid quantitative analysis of neutrophils in mouse cornea by flow cytometry

Objective

To provide a standard protocol for the rapid quantitative analysis of neutrophils in inflamed corneas with flow cytometry.

Methods

The corneal epithelium layer of 15 C57BL/6 mice (6-8 weeks old) was mechanically scraped off using a golf-like knife to generate a 2 mm wound region.The mouse corneas with intact limbus were cut out at 18 hours after abrasion.After mechanical shredding, the single cell suspension was obtained by collagenase I and DNase digestion.Then, the number of neutrophils in the corneal cells was sorted under the FACSCanto flow cytometer using the gate technique.Another 6 mice were taken and randomized into wounded group and normal group according to a random number table method, with 3 mice in each group.Corneal cell staining was performed using fluorescent-conjugated anti-mouse CD45, Ly6G, and CD11b antibodies.The number of neutrophils in the corneas of the two groups were enumerated and compared.The use and care of the animals complied with the Statement of the Association for Research in Vision and Ophthalmology (ARVO). The study protocol was approved by the Animal Ethics Committee of Medical College of Jinan University (No.JN-A-2002-01).

Results

A standard procedure for detecting neutrophils in the cornea by flow cytometry was established.The ratio of CD45⁺ cells in the total corneal tissue cell population was (20.93±1.72)%.The Ly6G⁺ and CD11b⁺ double positive neutrophil population was sorted in the wounded corneal cell population.The ratios of Ly6G⁺ and CD116⁺ cells in the CD45⁺ cells were (75.50±3.25)% and (93.40±4.53)%, respectively, and the ratio of the Ly6G⁺ and CD11b⁺ double positive neutrophils in the total number of CD45⁺ cells was (67.33±2.80)%.In addition, the number of neutrophils recruited to the cornea at 18 hours after corneal abrasion was (151.47±10.82)%, which was higher than (15.36±1.02)% in the normal cornea (t=21.689, P<0.01).

Conclusions

Flow cytometry can quickly and accurately quantitatively analyze the neutrophil population in the wounded cornea.It provides a rapid quantitative analysis method to further evaluate the changes of neutrophils in corneal inflammation caused by different reasons.

Cornea; Flow cytometry; Neutrophils