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. 2023 Oct 14;26(11):108213. doi: 10.1016/j.isci.2023.108213

Figure 7.

Figure 7

The inflammatory potential of myeloid cells found in LEW rats is greater than that found in DA rats

Myeloid cell inflammatory potential was evaluated after lipopolysaccharide (LPS) stimulation of freshly isolated liver-resident non-parenchymal cells. LPS-induced TNFα secretion was measured via intracellular cytokine staining (ICS). The non-parenchymal liver cell dissociate was obtained via a gentle enzymatic perfusion process and differential centrifugation. The resulting cells were plated in 12 well plates for 3.5 h before being stimulated for 6 h under a concentration of 1 ng/mL of LPS in the presence of 1:1000 concentration of Monensin and Brefeldin.

(A) Flow cytometry plots showing the gating strategy for macrophages.

(B) Percentage of TNFα+ secreting CD68+CD11b+ myeloid cells in the unstimulated control and stimulated conditions of Dark Agouti and Lewis macrophages.

(C) Summary graphs of Lewis versus Dark Agouti total TNFα as a percentage of CD68+CD11b+myeloid, (D) and of the mean fluorescence intensity (MFI) of Lewis vs. Dark Agouti TNFα.

(E) Representative flow cytometry plot of TNFα secretion patterns based on ITGAL subpopulations. (F) and summary graph ITGAL expressing CD68+CD11b+ myeloid subpopulations. Plotted are the values from all 4 experimental replicates. Statistical significance for ICS was determined using a non-parametric 2 tailed Mann-Whitney test. (n = 4) (G) Cytometric bead array (LEGENDplex) was performed to quantify the level of cytokines (TNFα, Il-18, CXCL1) on culture supernatants of enriched CD68+ myeloid cells after 24 h of stimulation in various LPS concentration conditions (0, 0.05, 0.1, 1, 10 ng/mL). Three technical replicates were used per animal. Statistical significance of the CBA was determined using a two-way ANOVA and Sidak’s multiple comparisons test (n = 3) Data are represented as mean ± SEM with each dot representing a single animal. (∗: p value <0.05, ∗∗: p value <0.01, ∗∗∗: p value <0.001,∗∗∗∗: p value <0.0001) DA: dark agouti, LEW: lewis, SSC-A: side scatter area, FSC-A: forward scatter area.