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. 2019 Oct 1;8:e41239. doi: 10.7554/eLife.41239

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© 2019, Simmons et al

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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Figure 2. — (A) FACS analysis of CD4⁺ and CD8⁺ SP T-cells (left) and mature rec. B-cells (right) of lymph fluid isolated from the cisterna chyli of Spns2^f/f and Lyve1;Spns2^Δ/Δ mice. (B) Experimental flow-chart of short-terming homing assays to quantify lymphocyte immigration into pLNs. (C) FACS analysis of total congenic CD45.1⁺ cells in pLNs two hours upon injection of WT splenocytes into Spns2^f/f and Lyve1;Spns2^Δ/Δ mice. (D) Light microscopy of frozen sections of pLNs of Spns2^f/f (left) and Lyve1;Spns2^Δ/Δ (right) mice for PNAd⁺ HEVs (blue) counterstained for collagen-IV⁺ (brown). (E) FACS analysis of total CD45^-/CD31⁺/PNAd⁺ high-endothelial cells isolated from pLNs of Spns2^f/f and Lyve1;Spns2^Δ/Δ mice. (F) Experimental flow-chart of homing assays to quantify lymphocyte egress from pLNs. (G) Total numbers of congenic eGFP⁺ cells in pLNs at 0 hr and 20 hr upon injection of anti-α₄ / anti-α_L antibodies into Spns2^f/f and Lyve1;Spns2^Δ/Δ mice. Each circle (A, C, E, G) represents an individual mouse; bars indicate the mean. Scale bars, 50 μm (D). **p<0.005; ***p<0.0005 (two-tailed unpaired Student’s t-test (A, C, E, G)). Data are representative for five mice per group pooled from two independent experiments (A) with n = 2 or n = 3 mice per group (A), for six mice per group (D) or are pooled from two (C, G) or three (E) independent experiments with n = 2, n = 3 or n = 4 mice per group.

Figure 2—figure supplement 1. — (A) FACS analysis of CD4⁺ and CD8⁺ SP T-cells (left) and mature rec. B-cells (right) of lymph fluid isolated from the cisterna chyli of Spns2^f/f and Lyve1;Spns2^Δ/Δ mice. (B) Experimental flow-chart of short-terming homing assays to quantify lymphocyte immigration into pLNs. (C) FACS analysis of total congenic CD45.1⁺ cells in pLNs two hours upon injection of WT splenocytes into Spns2^f/f and Lyve1;Spns2^Δ/Δ mice. (D) Light microscopy of frozen sections of pLNs of Spns2^f/f (left) and Lyve1;Spns2^Δ/Δ (right) mice for PNAd⁺ HEVs (blue) counterstained for collagen-IV⁺ (brown). (E) FACS analysis of total CD45^-/CD31⁺/PNAd⁺ high-endothelial cells isolated from pLNs of Spns2^f/f and Lyve1;Spns2^Δ/Δ mice. (F) Experimental flow-chart of homing assays to quantify lymphocyte egress from pLNs. (G) Total numbers of congenic eGFP⁺ cells in pLNs at 0 hr and 20 hr upon injection of anti-α₄ / anti-α_L antibodies into Spns2^f/f and Lyve1;Spns2^Δ/Δ mice. Each circle (A, C, E, G) represents an individual mouse; bars indicate the mean. Scale bars, 50 μm (D). **p<0.005; ***p<0.0005 (two-tailed unpaired Student’s t-test (A, C, E, G)). Data are representative for five mice per group pooled from two independent experiments (A) with n = 2 or n = 3 mice per group (A), for six mice per group (D) or are pooled from two (C, G) or three (E) independent experiments with n = 2, n = 3 or n = 4 mice per group.