flow cytometry cd3ε  (Cytek Biosciences)

Journal: Frontiers in Immunology

Article Title: LTr1 alleviates DSS-induced ulcerative colitis by modulating macrophages to inhibit M1 polarization and associated inflammatory responses

doi: 10.3389/fimmu.2025.1651922

Figure Lengend Snippet: Anti-inflammatory effects of LTr1 in DSS-induced colitis. (A) The representative images of the spleen and quantitative analysis of spleen weight. (B) ELISA analysis of IL-1β, IL-6, TNFα, and IL-12 in mice ocular blood serum. (C) mRNA expression levels of IL-1β, IL-6, TNFα, IL-12, IFN-γ, and IL-10 in colon tissue were determined by quantitative RT-PCR. (D) Immunohistochemical staining analysis of CD3, and F4/80 in colon tissue. Scale bars represent 100μm. All experiments were performed with n = 5 mice per group and presented as means ± SD. Statistical analysis was performed using one-way ANOVA followed by LSD multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The following antibodies were used for flow cytometry: CD3-Brilliant Violet 510 (eBiosciences, Cat# 464882), CD11b-Brilliant Violet 421 (Biolegend, Cat# 101235), CD19-Brilliant Violet 650 (BD Biosciences, Cat# 563235), CD45-FITC (Biolegend, Cat# 103108), F4/80-PE (BD Biosciences, Cat# 565410). iNOS-APC (Miltenyibiotec, Cat# 130-116-423), CD80-Brilliant Violet 711 (Biolegend, Cat# 104743), CD206-PE/Cyanine7 (Biolegend, Cat# 141720).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Immunohistochemical staining, Staining

Journal: Frontiers in Immunology

Article Title: LTr1 alleviates DSS-induced ulcerative colitis by modulating macrophages to inhibit M1 polarization and associated inflammatory responses

doi: 10.3389/fimmu.2025.1651922

Figure Lengend Snippet: LTr1 reduces DSS-induced macrophage infiltration and M1 Polarization. (A) The proportions of macrophages in the splenocytes and LNLs were determined by flow cytometry analysis. (B) Protein expression levels of iNOS and CD80 in macrophages of LNLs, along with the percentage of iNOS + or CD80 + macrophages as determined by flow cytometry. (C) mRNA expression levels of iNOS , CD80 , Arg1 , CD206 in colon tissue determined by quantitative RT-PCR. All experiments were performed with n = 5 mice per group and presented as means ± SD. Statistical analysis was performed using one-way ANOVA followed by LSD multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The following antibodies were used for flow cytometry: CD3-Brilliant Violet 510 (eBiosciences, Cat# 464882), CD11b-Brilliant Violet 421 (Biolegend, Cat# 101235), CD19-Brilliant Violet 650 (BD Biosciences, Cat# 563235), CD45-FITC (Biolegend, Cat# 103108), F4/80-PE (BD Biosciences, Cat# 565410). iNOS-APC (Miltenyibiotec, Cat# 130-116-423), CD80-Brilliant Violet 711 (Biolegend, Cat# 104743), CD206-PE/Cyanine7 (Biolegend, Cat# 141720).

Techniques: Flow Cytometry, Expressing, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: LTr1 alleviates DSS-induced ulcerative colitis by modulating macrophages to inhibit M1 polarization and associated inflammatory responses

doi: 10.3389/fimmu.2025.1651922

Figure Lengend Snippet: LTr1 directly acted on macrophages. (A) Cytotoxicity of LTr1 to RAW264.7 cell line was determined by CCK8 cell viability assay. (B) Protein expression levels of iNOS, CD80, and CD206 in RAW264.7 cell line was determined by flow cytometry. (C) ELISA analysis of IL-1β and IL-6 in cell culture medium. (D) mRNA expression levels of iNOS , IL-1β , and IL-6 in colon tissue determined by quantitative RT-PCR. Data were collected from at least three independent experiments and presented as means ± SD. All data statistical differences were evaluated using Permutation test and Bonferroni correction. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The following antibodies were used for flow cytometry: CD3-Brilliant Violet 510 (eBiosciences, Cat# 464882), CD11b-Brilliant Violet 421 (Biolegend, Cat# 101235), CD19-Brilliant Violet 650 (BD Biosciences, Cat# 563235), CD45-FITC (Biolegend, Cat# 103108), F4/80-PE (BD Biosciences, Cat# 565410). iNOS-APC (Miltenyibiotec, Cat# 130-116-423), CD80-Brilliant Violet 711 (Biolegend, Cat# 104743), CD206-PE/Cyanine7 (Biolegend, Cat# 141720).

Techniques: Viability Assay, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Cell Culture, Quantitative RT-PCR

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: CSPG4-specific CAR.CIK lymphocytes as a novel therapy for the treatment of multiple soft tissue sarcoma histotypes

doi: 10.1158/1078-0432.CCR-20-0357

Figure Lengend Snippet: CSPG4 mRNA expression in multiple STS histotypes (Leiomyosarcoma; Dedifferentiated Liposarcoma; Undifferentiated pleomorphic sarcoma (UPS), Malignant Fibrous Histiocytoma, High-Grade Spindle Cell Sarcoma; Myxofibrosarcoma; Malignant Peripheral Nerve Sheath Tumor; Synovial Sarcoma). CSPG4 expression in STS was comparable to that observed in melanoma. RNA-sequencing expression data were selected and downloaded from the cBioPortal of the TCGA Pan-Cancer Collections. RSEM expression values were plotted after Log2 transformation with 0.5 jittering on the x-axis, using Microsoft Excel®. (A). CSPG4 expression was confirmed in patient-derived STS cell lines of various histologic types by flow cytometry. A representative flow-cytometry histogram is reported for each STS. The M14 melanoma cell line that lacks CSPG4 expression and normal keratinocytes were used for comparison. Isotype controls are shown in grey (B). Grey histograms show the number of CSPG4 molecules expressed on the cell surface of various patient-derived STS cell lines quantified as the CSPG4-specific mAb-binding capacity (sABC) on a per cell basis (C).

Article Snippet: Flow cytometry Conjugated CD3, CD4, CD8, CD56, PD-1, CXCR3, CXCR4, and CCR7 mAbs (BD Pharmingen) and CD45RO, CD45RA, and CD62L mAbs (Miltenyi Biotec) were used to characterize lymphocytes.

Techniques: Expressing, RNA Sequencing, Transformation Assay, Derivative Assay, Flow Cytometry, Comparison, Binding Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: CSPG4-specific CAR.CIK lymphocytes as a novel therapy for the treatment of multiple soft tissue sarcoma histotypes

doi: 10.1158/1078-0432.CCR-20-0357

Figure Lengend Snippet: Schematic representation of the STS xenografts and treatment with CSPG4-CAR.CIK (red arrows) and NTD.CIK (blue arrows). Vehicle-treated mice were infused with PBS (grey arrows). CIK were infused intravenously (1x106 cells/infusion) twice a week for 2 weeks (A). Autologous CSPG4-CAR.CIK caused a significant delay of the growth of the S172 leiomyosarcoma (CSPG4=72% and CSPG4 density=262 molecule/cell) as compared to unmodified NTD.CIK or vehicle-treated mice (n=6; p<0.05) (B). Autologous CSPG4-CAR.CIK caused a significant delay of the growth of the HT1080 fibrosarcoma (CSPG4=23% and CSPG4 density=521 molecule/cell) as compared to unmodified NTD.CIK or vehicle-treated mice (n=3, p<0.001). (C). Autologous CSPG4-CAR.CIK effectively delayed the growth of S1 UPS (CSPG4=95% and CSPG4 density=499 molecule/cell) as compared with controls (n=3, p<0.0001). All results were analyzed by two-way ANOVA and the Bonferroni post-test; statistical significance is reported as * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001. (D). Tumor-homing of CSPG4-CAR.CIK and unmodified NTD.CIK was confirmed by IHC in explanted tumors using an anti-human CD3 antibody staining. Magnification: 40X; Scale bars: 50 μm (E). Apoptotic tumor cells were visualized by detecting cleaved caspase 3 by IHC in explanted tumors (F).

Article Snippet: Flow cytometry Conjugated CD3, CD4, CD8, CD56, PD-1, CXCR3, CXCR4, and CCR7 mAbs (BD Pharmingen) and CD45RO, CD45RA, and CD62L mAbs (Miltenyi Biotec) were used to characterize lymphocytes.

Techniques: Staining