Percentage of IL-17A⁺ ILC3 subsets among CD45⁺ immune cellsh Percentage of IL-22⁺ ILC3 subsets among CD45⁺ immune cells

Experimental Design
Core Objective: Flow cytometry was performed to evaluate the effect of RORγt⁺ cell-specific Atg5 knockout (autophagy deficiency) on the effector cytokine production of intestinal lamina propria group 3 innate lymphoid cells (ILC3s) in mice with necrotizing enterocolitis (NEC). Specifically, we quantified the proportion of IL-17A-producing and IL-22-producing cells within total ILC3s and double-negative (DN) ILC3 subsets, relative to total CD45⁺ immune cells.
Animal Model:Genetically modified mice on a C57BL/6 background were used in this study:
Atg5fl/fl mice: Littermate wild-type control mice with intact Atg5-mediated autophagy function in RORγt⁺ cells
RorccreAtg5fl/fl mice: RORγt⁺ cell-specific conditional Atg5 knockout mice, with autophagy deficiency specifically restricted to the ILC3 lineageAll mice were subjected to standardized experimental NEC induction prior to sample collection and flow cytometry detection.
Experimental Groups (2 groups total):
Control group: Atg5fl/fl mice with NEC induction (autophagy-intact ILC3s)
Knockout group: RorccreAtg5fl/fl mice with NEC induction (ILC3-specific autophagy deficiency)
Detection Targets:The proportion of cytokine-producing ILC3 subsets relative to total CD45⁺ immune cells in the intestinal lamina propria, including:
Percentage of IL-17A⁺ cells within total ILC3s among CD45⁺ cells
Percentage of IL-17A⁺ cells within DN ILC3s among CD45⁺ cells
Percentage of IL-22⁺ cells within total ILC3s among CD45⁺ cells
Percentage of IL-22⁺ cells within DN ILC3s among CD45⁺ cells
Biological Replicates: 6 biological replicates (individual mice) per group
Flow cytometry data were acquired using a CytoFLEX S flow cytometer (Beckman Coulter). The following fluorochrome-conjugated antibodies were used for cell surface and intranuclear staining in this study:NKp46-FITC, Lin-APC-Cy7, live/dead-BV510, CD4-Pacific Blue (PB), CD90.2-PE-Cy7, IL17A/IL22-PE, CCR6-APC, and RORγt-PerCP-Cy5.5.
Statistical Analysis: Unpaired two-tailed Student's t-test was applied for comparisons between the two independent groups. Statistical significance was defined as P < 0.05.