mouse anti icam1 bba3  (R&D Systems)

Journal: Oncotarget

Article Title: A novel immune resistance mechanism of melanoma cells controlled by the ADAR1 enzyme

doi:

Figure Lengend Snippet: A. ICAM1 protein expression in ADAR1-p110 (dotted line) and Mock cells (black line) was analyzed by extracellular flow cytometry staining using ICAM1 monoclonal antibody. B. LFA-1 protein expression in TIL52, TIL14 and JKF6 was analyzed by extracellular flow cytometry staining using human LFA-1 alpha antibody. C. ADAR1-p110 and Mock cells were incubated with different concentrations of IgG1 control (isotype) or ICAM1 antibody. After 1 h, cells were co-incubated with JKF6 for 9 h (E:T - 15:1). Specific lysis of melanoma cells was assessed by LDH release. Experiments were performed three times in triplicates. Flow cytometry figures show one representative experiment.

Article Snippet: For blocking assays, target cells were pre-incubated for 1 h on ice with different concentrations of anti-Human ICAM1 monoclonal mouse IgG1 antibody (R&D Systems, cat#BBA3) or mouse IgG1 isotype control (BioXCell, cat#BE0083), followed by 9 h cytotoxicity assays.

Techniques: Expressing, Flow Cytometry, Staining, Incubation, Control, Lysis

Journal: Oncotarget

Article Title: A novel immune resistance mechanism of melanoma cells controlled by the ADAR1 enzyme

doi:

Figure Lengend Snippet: A–B. 624mel cells were transfected with miR-222 precursor, mir-221 precursor or control (pQCXIP) plasmid. Expression levels were assessed by qRT-PCR and normalized to U6 expression. C–D. ICAM1 mRNA levels in miR-222 OX, miR-221 OX and pQCXIP cells were assessed by qRT-PCR and normalized to GAPDH expression. E–F. ICAM1 protein expression in miR-222 OX (dotted line) (E), miR-221 OX (dotted line) (F) and pQCXIP (black line) cells was analyzed by extracellular flow cytometry staining. G–H. miR-222 OX or miR-221 OX cells and pQCXIP cells were co-incubated in different E:T ratios with JKF6 or TIL52 for 5 h or overnight. Specific lysis of melanoma cells was assessed by flow cytometry or LDH release. I. 293T cells were co-transfected with miR-222, mir-221 or control (pQCXIP empty vector) constructs and with ICAM1 UTR or ICAM1 UTR MUT which is mutated at the predicted binding site of miR-221 and miR-222. Dual luciferase assay was carried out and Renilla luciferase activity was measured and normalized to the firefly constitutive luciferase activity. Relative Luciferase activity was normalized to the Luciferase activity of control vector. NS denotes “not significant”. Experiment was performed three times in sixplicates. J. miR-222 OX and pQCXIP cells were incubated with different concentrations of IgG1 control (isotype) or ICAM1 antibody. After 1 h, cells were co-incubated with JKF6 for 9 h (E:T - 15:1). Specific lysis of melanoma cells was assessed by LDH release. Experiments were performed three times in triplicates. Flow cytometry figures show one representative experiment.

Article Snippet: For blocking assays, target cells were pre-incubated for 1 h on ice with different concentrations of anti-Human ICAM1 monoclonal mouse IgG1 antibody (R&D Systems, cat#BBA3) or mouse IgG1 isotype control (BioXCell, cat#BE0083), followed by 9 h cytotoxicity assays.

Techniques: Transfection, Control, Plasmid Preparation, Expressing, Quantitative RT-PCR, Flow Cytometry, Staining, Incubation, Lysis, Construct, Binding Assay, Luciferase, Activity Assay

Journal: Oncotarget

Article Title: A novel immune resistance mechanism of melanoma cells controlled by the ADAR1 enzyme

doi:

Figure Lengend Snippet: A. ADAR1 His-tagged mRNA levels were assessed by qRT-PCR and normalized to GAPDH expression. Protein expression of ADAR1 His-tagged constructs was determined by WB using ADAR1 polyclonal antibody and polyHistidine monoclonal antibody (cut from different regions in the same membrane). B. Expression levels of hsa-miR-222 in ΔCAT-S and Mock cells were assessed by qRT-PCR and normalized to U6 expression. C. ICAM1 protein expression in ΔCAT-S (dotted line) and Mock cells (black line) was analyzed by extracellular flow cytometry staining. D. ΔCAT-S and Mock cells were co-incubated with JKF6 for 5 h (E:T - 15:1). Specific lysis of melanoma cells was assessed by flow cytometry. Experiments were performed three times in triplicates. WB and flow cytometry figures show one representative experiment.

Article Snippet: For blocking assays, target cells were pre-incubated for 1 h on ice with different concentrations of anti-Human ICAM1 monoclonal mouse IgG1 antibody (R&D Systems, cat#BBA3) or mouse IgG1 isotype control (BioXCell, cat#BE0083), followed by 9 h cytotoxicity assays.

Techniques: Quantitative RT-PCR, Expressing, Construct, Membrane, Flow Cytometry, Staining, Incubation, Lysis