pe cy7 tonbo biosciences 60 1886 u100 2 43 cd8b pacific blue biolegend 140414  (Cytek Biosciences)

Journal: bioRxiv

Article Title: TGF-β induces an atypical EMT to evade immune mechanosurveillance in lung adenocarcinoma dormant metastasis

doi: 10.1101/2024.10.15.618357

Figure Lengend Snippet: a , 3D reconstructed images of H2087-LCC and M802T4-LCC cells disseminated in brain parenchyma at indicated time point after intracardiac inoculation into athymic mice and B6(Cg)-Tyrc-2J/J (B6-albino) mice, respectively. Cancer cells were visualized by GFP IF (green) and brain capillaries by CD31 IF (magenta). Scale bar: 10 μm. b , Quantification of elongated, spheroidal, and microclustered cancer cells (fewer than 10 cells) in A. n=33 and 18 for H2087-LCC cells on day7 and day 49 respectively. n=253 and 135 for M802T4-LCC cells on day 7 and day 49 respectively. Chi-square test. c , Representative images of H2087-LCC and M802T4-LCC cells expressing a TGF-β reporter in the lungs of athymic mice and B6129SF1/J mice, respectively, 7 weeks after intravenous injection. Cancer cells were visualized by GFP IF (green), the TGF-β mCherry reporter activity in red, Ki67 proliferation marker in white, and DAPI in blue. Scale bar: 20 μm. d , Quantification of TGF-β mCherry reporter and Ki67 expression in H2087-LCC (n=129) and M802T4-LCC (n=63) cells in the lungs of athymic and B6129SF1/J mice, respectively, 7 weeks after intravenous injection. e and f , Representative IF images of H2087-LCC in the lungs of athymic mice 7 weeks after intravenous injection. GFP + cancer cells (green), TGF-β mCherry reporter (red), E-cadherin or FN1 (white), and DAPI (blue). g , Quantification of TGF-β mCherry reporter and E-cadherin (n=87) or FN1 (n=53) expression in disseminated H2087-LCC cells in the lungs of athymic mice. h and i , Representative IF images of M802T4-LCC in the lungs of B6129SF1/J mice 7 weeks after intravenous injection. GFP + cancer cells (green), TGF-β mCherry reporter (red), E-cadherin (white), and DAPI (blue). Scale bar: 20 μm. j , Quantification of TGF-β mCherry reporter and E-cadherin (n=71) or FN1 (n=51) expression in M802T4-LCC in the lungs of B6129SF1/J mice 7 weeks after intravenous injection. k-m , Schematic representation of the experimental design, created with Biorender.com (k), and tracking of wild-type and Tgfbr2 KO M802T4-LCC cells intravenously inoculated in B6129SF1/J mice followed by antibody-mediated depletion of NK, CD4+ and CD8+ T cells from day 3 (l) or day 35 (m) after inoculation. n=6 or 7 mice per group.

Article Snippet: To validate the depletion of each immune population, peripheral blood was collected and stained with violetFluor 450 Anti-Mouse CD45 (30-F11) (Tonbo Biosciences, Cat# 75-0451-U100), FITC Anti-Mouse CD4 (GK1.5) (Tonbo Biosciences, Cat# 35-0041-U100), PE-Cy7 Anti-Mouse CD8a (53-6.7) (Tonbo Biosciences, Cat#60-0081-U100), APC Anti-Mouse NK1.1 (CD161) (PK136) (Tonbo Biosciences, Cat# 20-5941-U100), followed by flow cytometric analysis on an LSRFortessa (BD Biosciences) instrument.

Techniques: Expressing, Injection, Activity Assay, Marker

Journal: bioRxiv

Article Title: Assessing a Role for Sarcospan in Immune Function

doi: 10.1101/2024.03.31.587501

Figure Lengend Snippet: Immunophenotype Results for Male and Female Sarcospan-Deficient Mice. Flow cytometry was used to determine the % immune cell populations in splenocyte suspensions obtained from WT and SSPN -/- male and female mice. In (A) T cells, (B) plasmacytoid dendritic cells (pDCs), (C) dendritic cells (DCs), and (G) natural killer NK1.1 + cells. Representative flow spectra are shown in (D) CD4+ and CD8+ T cells (E) pDCs, and (F) DCs. Genotype differences in the male and female mouse groups were assessed separately using the two-tailed Student’s t-test. Data shown as individual values and considered significant if p ≥ 0.05. Significant differences indicated by * for 0.05 and ** for 0.001.

Article Snippet: Cells were incubated with anti-mouse primary antibodies to assess immune populations with the following conjugated antibodies: B220-APC (Tonbo Biosciences, San Diego, CA; clone RA3-6B2), CD19-PE/Dazzle594 (BioLegend, San Diego, CA; clone 6D5), CD3-AlexaFluor488 (BioLegend, clone 17A2), CD4-violetFluor450 (Tonbo Biosciences, clone GK1.5), CD8-PE/Cy7 (Tonbo Biosciences, clone 53-6.7), NK1.1-PE (Tonbo Biosciences, clone PK136), CD335/NKp46-APC (BioLegend, clone 29A1.4), CD11b-APC/Cy7 (Tonbo Biosciences, clone M1/70), CD11c-PerCp/Cy5.5 (Tonbo Biosciences, clone N418), Ly6G-PE (Tonbo Biosciences, clone RB6-8C5), and F4/80-FITC (Tonbo Biosciences, clone BM8.1,).

Techniques: Flow Cytometry, Two Tailed Test, IF-P