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. 2020 Mar 26;8(1):e000172. doi: 10.1136/jitc-2019-000172

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© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See http://creativecommons.org/licenses/by-nc/4.0/.

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Generation and immunophenotypical characterization of shCD6LckEμTg mice. (A) Schematic representation of the transgene coding for soluble human CD6 (shCD6). (B) PCR screening of genomic DNA from homozygous shCD6LckEµTg and non-transgenic (NonTg) mice. Bands correspond to transgene (Tg) and internal PCR control (C). (C) ELISA quantification of plasma shCD6 levels from NonTg (-/-), and heterozygous (-/+) and homozygous (+/+) shCD6EμLckTg (Tg) mice. Represented are cumulative data from two independent experiments, expressed as mean±SEM. ***p<0.001; two-tailed Student’s t-test. (D) Immunophenotypical characterization of primary lymphoid organs from shCD6LckEμTg mice. Left: total lymphoid cell numbers from thymus and bone marrow of shCD6LckEμTg (n=21 and n=13, respectively) and NonTg (n=15 and n=16, respectively) mice. Middle: percentage of CD24^lowBP1^- (pre-pro B cells), CD24⁺BP1^- (early pro B cells), CD24⁺BP1⁺ (late pro B cells) and CD24^highBP1⁺ (pre B cells) within gated CD43⁺B220⁺ bone marrow cells from shCD6LckEμTg and NonTg mice. Right: percentage of CD4^-CD8^- (DN), CD4⁺CD8⁺ (DP), CD4⁺CD8^- (CD4SP) and CD4^-CD8⁺ (CD8SP) thymocytes from shCD6LckEμTg and NonTg mice. Represented are cumulative data from at least two independent experiments, expressed as mean±SEM. *p<0.05; **p<0.01; two-tailed Student’s t-test. (E) Immunophenotypical characterization of secondary lymphoid organs from shCD6LckEμTg mice. Left: total lymphoid cell numbers in spleen, and lymph node (LN) from shCD6LckEμTg (n=50 and n=23, respectively) and NonTg (n=61 and n=28, respectively) mice. Right: percentage of viable cells (7AAD^- AnnexinV^-) from spleen and LN of shCD6LckEμTg and NonTg mice. One of two independent experiments is shown. (F) In vivo Bromodeoxyuridine (BrdU)-incorporation assays in secondary lymphoid organs from shCD6LckEμTg mice. Percentage of spleen (left) and LN (right) BrdU⁺ lymphoid cells represented as total cells and as cells gated on CD19⁺, CD4⁺ and CD8⁺. Represented are cumulative data from at least two independent experiments, expressed as mean±SEM. *p<0.05; **p<0.01; ***p<0.001; two-tailed Student’s t-test.