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. Author manuscript; available in PMC: 2021 Mar 23.
Published in final edited form as: J Leukoc Biol. 2020 May 9;107(6):883–892. doi: 10.1002/JLB.4HI0420-098R

Figure 2. Nonclassical monocytes lacking Kindlin-3 fail to enter lung tissue and do not surround invading metastases.

Figure 2.

A) Snapshots of lung intravital imaging (Leica SP8, 25× 0.8 NA objective) in CX3CR1gfp/+Kindlin-3fl/flMx1-cre- (WT) or cre+ (K-KO) mice 30 minutes after i.v. injection of 3×105 B16F10-RFP cells. Scale bar = 50 uM. B) Analysis of intravital microscopy imaging from A), using Imaris software to detect the average number of GFP+ monocytes per for every frame of the recording within 30 μM of B16F10-RFP cells. n = 3 mice per group, independent experiments. C) Number of patrolling nonclassical monocytes per FOV from A). D) Flow cytometry gating for selection of vascular (CD45v) or interstitial (CD45i) leukocytes, followed by monocyte gating using CD115 and CD11b markers. Lungs were mechanically dissociated so as not to lose CD115 expression. E) Ly6C− monocyte frequencies within interstitial lung tissues out of total CD45+ live cells. n = 6–7 per group. F) Ly6C− monocyte frequencies within the vascular niches of the lung tissue out of total CD45+ live cells. n=6–7 mice per group. G) 3×105 B16F10 cells were labeled with CellTrace Violet and injected i.v. into DsRed:K-KO BMT mice. 16 hours later, classical (Ly6C+) and nonclassical (Ly6C−) monocytes from WT (DsRed) donors or K-KO donors within the same mice were assessed for uptake of B16F10-violet particles per 105 CD45+ cells in the lung. n = 8 mice per group. Kolmogorov-Smirnov nonparametric test for single comparisons, Kruskal-Wallis nonparametric test for multiple comparisons. ns = not significant, *p<0.05, **p<0.01, ****p<0.0001.