Evaluation of lipid metabolism in ILC3 cells from Atg5-deficient mice

Experimental Design
Core Objective: Flow cytometry was performed to evaluate the effects of RORγt⁺ cell-specific Atg5 knockout (autophagy deficiency) on cellular lipid metabolism and functional markers in the intestinal lamina propria of mice with necrotizing enterocolitis (NEC). Specifically, we quantified the BODIPY-derived mean fluorescence intensity (MFI) as an indicator of intracellular lipid storage, along with the metabolic and functional markers C11-BODIPY, DCFDA, and FCL16, to assess lipid dynamics and oxidative stress in RorccreAtg5fl/fl mice compared to wild-type controls.
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 lineage
All mice were subjected to standardized experimental NEC induction prior to sample collection for flow cytometry detection.
Experimental Groups (2 groups total, fully matched to the figure):
Atg5fl/fl group: Control group (NEC-induced mice with autophagy-intact RORγt⁺ cells)
RorccreAtg5fl/fl group: Autophagy-deficient group (NEC-induced mice with RORγt⁺ cell-specific Atg5 deletion)
Detection Targets (fully matched to the figure axes):Flow cytometric measurement of mean fluorescence intensity (MFI) reflecting intracellular lipid metabolism and stress in immune cells, including:
BODIPY MFI (*10⁴): Assessment of intracellular lipid accumulation
C11-BODIPY MFI (*10³): Evaluation of lipid peroxidation and membrane lipid dynamics
DCFDA MFI (*10³): Assessment of intracellular reactive oxygen species (ROS) levels
FCL16 MFI (*10⁵): Analysis of fatty acid uptake or lipid trafficking
Biological Replicates: 5 biological replicates (individual mice) per group
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.