anti-myc tag antibody, mouse mab  (Sino Biological)

Journal: eLife

Article Title: Layilin regulates Treg motility and suppressive capacity in skin

doi: 10.7554/eLife.105277

Figure Lengend Snippet: ( A ) Schematic experimental design strategy starting with the isolation of human Tregs from peripheral blood, activated and expanded using anti-CD3/anti-CD28 stimulation and IL-2 for 12 days. We deleted LAYN using CRISPR/Cas9, and after 4 days, cells were used for the experiment. ( B ) Frequency of LAYN + Tregs gated on live CD8 - CD4 + CD25 + FOXP3 + cells and representative expression of layilin. n=6 donors from two independent experiments. Significance was defined by a two‐tailed Student’s t ‐test. *p<0.05. ( C ) Frequencies (on the left) and mean fluorescence intensity (MFI, on the right) of sg- LAYN and sg-CTRL Tregs gated on live CD8 - CD4 + CD25 + FOXP3 + cells expressing the different activation Treg markers listed on the x-axis. The top-row graphs show results from cells stimulated with anti-CD3/anti-CD28; the bottom graphs show results obtained from cells without anti-CD3/anti-CD28 stimulation. n=6 donors from two independent experiments. Significance was defined by using a multiple-paired t ‐test. ( D ) Proliferation percentage measured using cell trace violet for CD4 + T responder (left panel) or CD8 + T responder (right panel) after 3 days of suppression assay. Results are presented in the x-axis, and the different ratios of sg- LAYN and sg-CTRL Tregs and T responders used for the assay represent two independent experiments. Significance was defined by two‐way ANOVA, followed by Tukey’s multiple-comparisons test.

Article Snippet: The following anti-human antibodies and clones were used: layilin (clone #07, Sino Biological Cat# 10208-MM07, RRID: AB_2860113 ), PE-Streptavidin, CD3 (UCHT1), CD4 (SK3), CD8 (SK1), CD45RO (UCHL1), FoxP3 (PCH101), CD25 (M-A251), CTLA4 (14D3), ICOS (ISA-3), CD27 (O323), CD11c (3.9), HLA-DR (L243), CD137 (4B4-1), PD1 (EH12.2H7), Ki67 (EH12.2H7), LAG3 (11C3C65), CD38 (HIT2), CD62L (DREG56), CD69 (FN50), CD127 (eBioRDR5), GATA3 (L50-823), GZMB (GB11), and HELIOS (22F6).

Techniques: Isolation, CRISPR, Expressing, Two Tailed Test, Fluorescence, Activation Assay, Suppression Assay

Journal: eLife

Article Title: Layilin regulates Treg motility and suppressive capacity in skin

doi: 10.7554/eLife.105277

Figure Lengend Snippet: ( A ) The experimental design strategy starts with the isolation of human Tregs from peripheral blood, which were activated and expanded using anti-CD3/anti-CD28 stimulation and IL-2 for 12 days. We deleted LAYN using a CRISPR/Cas9; after 4 days, cells were sorted by flow cytometry based on LAYN expression, as represented by the representative histogram of LAYN expression on sgCtrl (stained with or without primary antibody detecting layilin followed by streptavidin-PE staining) or sg- LAYN Tregs, to obtain a pure population of LAYN + or LAYN - Tregs. After sorting, cells were placed in culture for follow-up experiments presented in panels (B), (C), and (F). ( B ) Volcano plot of differential genes expressed in LAYN + (on the left) or LAYN - (on the right) Tregs put in culture for 24 hours in the presence of anti-CD3/anti-CD28. Red dots represent differentially expressed genes, and black dots are not differentially expressed ones. n=4 paired donors for each group. ( C ) Top deregulated pathways obtained from a gene set enrichment analysis comparing LAYN + to LAYN - Tregs. The top axis and bar plot represent the normalized enrichment score (NES). The bottom axis and dot represent the adjusted p-value associated with pathways enrichment. n=4 paired donors for each group. ( D ) Ratio of m24 + to total CD18 + Tregs gated on live CD8 - CD4 + CD25 + FOXP3 + cells after 3 days culture with or without anti-CD3/anti-CD28 stimulation and in the presence of a layilin-crosslinking antibody as described on the x-axis for 20 minutes during LFA-1 activation staining. n=3 donors. Significance was defined by one‐way ANOVA, followed by Dunnett’s multiple-comparisons test. *p<0.05, ***p<0.001. ( E ) Frequency of m24 + sg- LAYN and sg-CTRL Tregs gated on live CD8 - CD4 + CD25 + FOXP3 + cells after 24 hours of culture in a non-coated plate or collagen IV-coated plate. n=6 donors from two independent experiments. Significance was defined by two‐way ANOVA, followed by Sidak’s multiple-comparisons test. *p<0.05. ( F ) Numbers of cells attached to the bottom of the well are measured by the cell titer blue (expressed by relative fluorescence unit). n=7 donors from two independent experiments. Significance was defined by two‐way ANOVA. ns, nonsignificant, ***p<0.001.

Article Snippet: The following anti-human antibodies and clones were used: layilin (clone #07, Sino Biological Cat# 10208-MM07, RRID: AB_2860113 ), PE-Streptavidin, CD3 (UCHT1), CD4 (SK3), CD8 (SK1), CD45RO (UCHL1), FoxP3 (PCH101), CD25 (M-A251), CTLA4 (14D3), ICOS (ISA-3), CD27 (O323), CD11c (3.9), HLA-DR (L243), CD137 (4B4-1), PD1 (EH12.2H7), Ki67 (EH12.2H7), LAG3 (11C3C65), CD38 (HIT2), CD62L (DREG56), CD69 (FN50), CD127 (eBioRDR5), GATA3 (L50-823), GZMB (GB11), and HELIOS (22F6).

Techniques: Isolation, CRISPR, Flow Cytometry, Expressing, Staining, Activation Assay, Fluorescence

Journal: eLife

Article Title: Layilin regulates Treg motility and suppressive capacity in skin

doi: 10.7554/eLife.105277

Figure Lengend Snippet: ( A ) Quality check of bulk RNAseq representing total counts for each sample (left panel) and LAYN count for each sample demonstrating CRISPR efficiency (right panel). n=4 paired donors for each group. ( B ) On the left panel, we represent the frequency of total m24+ regulatory T cells (Tregs) gated on live CD8-CD4+CD25+FOXP3 + cells after 3 days of culture with or without anti-CD3/anti-CD28 stimulation and in the presence of a layilin-crosslinking antibody as described on the x-axis for 20 minutes during lymphocyte function-associated antigen-1 (LFA-1) activation staining. The right panel represents the frequency of CD18 + Tregs. n=3 donors. Significance was defined by one‐way ANOVA, followed by Dunnett’s multiple-comparisons test. *p<0.05, ***p<0.001. ( C ) Frequency of LAYN + Tregs gated on live CD8 - CD4 + CD25 + FOXP3 + cells to measure CRISPR efficiency after 24 hours of culture in a non-coated plate or collagen IV-coated plate. n=6 donors from two independent experiments.

Article Snippet: The following anti-human antibodies and clones were used: layilin (clone #07, Sino Biological Cat# 10208-MM07, RRID: AB_2860113 ), PE-Streptavidin, CD3 (UCHT1), CD4 (SK3), CD8 (SK1), CD45RO (UCHL1), FoxP3 (PCH101), CD25 (M-A251), CTLA4 (14D3), ICOS (ISA-3), CD27 (O323), CD11c (3.9), HLA-DR (L243), CD137 (4B4-1), PD1 (EH12.2H7), Ki67 (EH12.2H7), LAG3 (11C3C65), CD38 (HIT2), CD62L (DREG56), CD69 (FN50), CD127 (eBioRDR5), GATA3 (L50-823), GZMB (GB11), and HELIOS (22F6).

Techniques: CRISPR, Activation Assay, Staining