Comparison of methods for the preparation of single-cell suspensions of rat retina and immunophenotyping by flow cytometry.

BACKGROUND

Although the eye has traditionally been considered an immune-privileged organ, progressive studies have re-evaluated the role of the immune response in retinopathy. The application of flow cytometry for immunophenotyping analysis of retinal single-cell suspensions has gradually attracted attention.

NEW METHODS

We dissociated the retinal tissue using trypsin digestion, papain digestion, mechanical grinding treatment and liberase + DNase Ⅰ digestion, respectively. Subsequently we assessed the quality of cell suspension (clumping rate, concentration and viability) with two different dyes, Trypan Blue (TPB) and Acridine Orange/Propidium Iodide (AO/PI). We distinguished T cells and their sub-populations by flow cytometry. Furthermore, we analyzed their immune responses to evaluate those four methods for preparing retinal single-cell suspension.

RESULTS

The AO/PI staining method enables a rapid and precise evaluation of cell quality of retinal single cell suspensions, while TPB staining has limitations. Flow cytometric analysis showed that single cell suspensions dispersed with papain and trypsin exhibited reduced cell adhesion. However, trypsin digestion may affect antibody binding. The mechanical grinding treatment reduced cell yield and was prone to double-positivity. The number of cells within the cell-circle gate is significantly limited in the Liberase + DNase I digestion method.

COMPARISON WITH EXISTING METHODS

The AO/PI staining method was employed to assess the quality of retinal single-cell suspensions. T cells and their subpopulations in retinal tissues were analyzed by flow cytometry. These results were integrated to evaluate the optimal preparation protocol for retinal single-cell suspensions.

CONCLUSIONS

Papain digestion is a superior method for preparing retinal single-cell suspensions.