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. 2021 Mar 26;10:e65412. doi: 10.7554/eLife.65412

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© 2021, Sheng 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 1. — (A) Representative flow cytometry dot plots for LC characterization in the epidermis. Cells from epidermis were first gated for FSC, SSC, and CD45 (not shown). Then, CD45⁺ cells were analysed for CD11b and F4/80 expression. The CD11b^hiF4/80^hi cell fraction was further analysed for CD207 and CD326 expression to identify classical bona fide LCs. (B) Representative flow cytometry dot plots for dermal LC and DC subpopulations. Isolated dermis cells were first gated for FSC, SSC, and CD45 (not shown). CD45⁺ cells were then analysed for CD11b and F4/80 expression. The CD11b^hi F4/80^hi fraction contained classical CD207⁺CD326⁺ LCs. The remaining cells were gated for CD11c⁺MHC II⁺ DCs and separated into three subsets based on CD103 and CD11b expression: CD103⁺CD11b^- cells (labelled cDC1), CD103^-CD11b^hi DCs (labelled CD11b^hi), and CD11b^low/neg. CD207 and CD326 expression was detectable on cDC1 but not CD11b^hi DCs, whereas CD11b^low cells were further separated into CD207^-CD326^- (labelled triple negative [TN]) and CD207⁺CD326⁺ (labelled LC^like). (C) Representative flow cytometry dot plots for cutaneous DC subpopulations in auricular skin-draining LNs. LN cells were first gated for FSC and SSC to exclude small lymphocytes before F4/80 and CD11b staining. The cell fraction excluding F4/80^hi/CD11b^hi cells was separated by CD11c and MHCII. CD11c^hiMHCII^hi migratory DCs were gated and analysed for CD103, CD11b, CD207, and CD326 expression. Four subsets were detected: CD103⁺CD11b^-CD207⁺CD326⁺ (cDC1), CD103^-CD11b^lowCD207^-CD326^- (TN), CD103^-CD11b^lowCD207⁺CD326⁺ (LC^like), and CD103^-CD11b^hiCD207^-CD326^-/+(CD11b^hi). (D) Frequency of each DC subpopulation (LC, cDC1, LC^like, TN, and CD11b^hi) present in epidermis, dermis, and cutaneous lymph node (LN), respectively.

Figure 1—figure supplement 1. — (A) Representative flow cytometry dot plots for LC characterization in the epidermis. Cells from epidermis were first gated for FSC, SSC, and CD45 (not shown). Then, CD45⁺ cells were analysed for CD11b and F4/80 expression. The CD11b^hiF4/80^hi cell fraction was further analysed for CD207 and CD326 expression to identify classical bona fide LCs. (B) Representative flow cytometry dot plots for dermal LC and DC subpopulations. Isolated dermis cells were first gated for FSC, SSC, and CD45 (not shown). CD45⁺ cells were then analysed for CD11b and F4/80 expression. The CD11b^hi F4/80^hi fraction contained classical CD207⁺CD326⁺ LCs. The remaining cells were gated for CD11c⁺MHC II⁺ DCs and separated into three subsets based on CD103 and CD11b expression: CD103⁺CD11b^- cells (labelled cDC1), CD103^-CD11b^hi DCs (labelled CD11b^hi), and CD11b^low/neg. CD207 and CD326 expression was detectable on cDC1 but not CD11b^hi DCs, whereas CD11b^low cells were further separated into CD207^-CD326^- (labelled triple negative [TN]) and CD207⁺CD326⁺ (labelled LC^like). (C) Representative flow cytometry dot plots for cutaneous DC subpopulations in auricular skin-draining LNs. LN cells were first gated for FSC and SSC to exclude small lymphocytes before F4/80 and CD11b staining. The cell fraction excluding F4/80^hi/CD11b^hi cells was separated by CD11c and MHCII. CD11c^hiMHCII^hi migratory DCs were gated and analysed for CD103, CD11b, CD207, and CD326 expression. Four subsets were detected: CD103⁺CD11b^-CD207⁺CD326⁺ (cDC1), CD103^-CD11b^lowCD207^-CD326^- (TN), CD103^-CD11b^lowCD207⁺CD326⁺ (LC^like), and CD103^-CD11b^hiCD207^-CD326^-/+(CD11b^hi). (D) Frequency of each DC subpopulation (LC, cDC1, LC^like, TN, and CD11b^hi) present in epidermis, dermis, and cutaneous lymph node (LN), respectively.