Figure 3: Intraperitoneal delivery of tamoxifen in peanut oil results in marked increases in macrophage autofluorescence.

Between 6–8 weeks of age, Myh11-Cre^ERT2eYFP or Myh11-Dre^ERT2tdTom mice received either 10 tamoxifen injections in peanut oil (Tmx inj), or pure peanut oil injections (Peanut Oil inj), or tamoxifen-containing diet (Tmx. Diet), or nothing (No injection, negative control) followed by a 2-week washout period. The mice were fed a normal diet or DIO diet for indicated times (W – weeks), tissues were harvested and epididymal adipose tissue SVF cells were subjected to flow cytometry. (A) Representative plots showing the frequency of CD45⁺ within eYFP (Myh11-Cre^ERT2eYFP panels) or tdTomato (Myh11-Dre^ERT2tdTom panels) gates based on eYFP or tdTomato FMOs. The eYFP and tdTomato FMO gates were set using SVF cells of epididymal adipose tissue from Myh11-Cre^ERT2eYFP or Myh11-Dre^ERT2tdTom control mice that received neither injections nor tamoxifen in the diet. (B) Quantification of flow cytometric analyses (Mean ± SEM) using different tamoxifen delivery methods and reporter lines. Myh11-Cre^ERT2eYFP (SMC-P-eYFP) mouse line – Tamoxifen injection in peanut oil (2W normal diet n = 12, 6W normal diet n = 10, 6W DIO n = 19), Tamoxifen Diet (normal diet (2W) n = 7, normal diet (6W) n = 3, DIO (6W) n = 4); Myh11-Dre^ERT2tdTom (SMC-P tdTom) mouse line – Tamoxifen injections in peanut oil (normal diet (4W) n = 6), Tamoxifen Diet (normal diet (4W) n = 5). (C) Epididymal adipose tissue SVF cells were stained with antibodies and subjected to ImageStream analysis. Live single cells excluding debris were gated for analysis. Representative cells were picked to illustrate differences in eYFP signals among single nuclei cells. Columns show endogenous eYFP signal (green), staining for GFP (anti-GFP), CD45, Viability dye, CD11b, DAPI, and F4/80. Side scatter (SSC) indicates granularity of the cells. (D) Myh11-Cre^ERT2eYFP mice received 10 tamoxifen injections in peanut oil or were fed tamoxifen in normal diet for 2 weeks followed by a 2-week washout period. Mice were fed a normal diet and euthanized at 12 weeks of age and tissues were harvested. Blood samples were stained with antibodies and subjected to flow cytometry. Circulating blood cell types are gated as follows: live/singlets/scatter gates were initially applied to remove dead cells, doublets and debris. CD45⁺ cells were gated for CD3⁺, then frequency of CD4 or CD8 single positive cells plotted. From the same CD45⁺ gate, CD11b⁺ cells are gated and plotted. From the CD11b⁺ gate the frequency of Ly6c^hi monocytes are plotted as well as Ly6G⁺ neutrophils (mean ± SEM). (E) Similar to (D), Epididymal adipose tissue SVF cell suspensions from the same mice were harvested and stained for flow cytometry. The percentage of CD45⁺ cells (mean ± SEM) among live and single cells are indicated, depicting the increased abundance of immune cells following peanut oil injection as compared to tamoxifen diet fed mice. (B, D, and E) P values were determined using an ordinary Two-way ANOVA with alpha=0.05 followed by Sidak’s multiple comparisons post-test.