Journal: Viruses

Article Title: Constructing TC-1-GLUC-LMP2 Model Tumor Cells to Evaluate the Anti-Tumor Effects of LMP2-Related Vaccines

doi: 10.3390/v10040145

Figure Lengend Snippet: Schematic demonstrating TC-1-GLUC-LMP2 cell construction. Latent membrane protein 2 ( LMP2 ) genes, internal ribosome entry site ( IRES ) and Gaussia luciferase ( GLuc ) sequences were inserted into TC-1 cells.

Article Snippet: Membranes were incubated with EBV LMP2 monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA, #sc-101314) diluted 1:500 in 5% skimmed milk, and a horseradish peroxidase-conjugated goat anti-rat IgG (ZSGB-BIO, Beijing, China, #ZB-2307) diluted 1:1000 was used as the secondary antibody.

Techniques: Membrane, Luciferase

Journal: Viruses

Article Title: Constructing TC-1-GLUC-LMP2 Model Tumor Cells to Evaluate the Anti-Tumor Effects of LMP2-Related Vaccines

doi: 10.3390/v10040145

Figure Lengend Snippet: The LMP2 and GLuc detection result in TC-1-GLUC-LMP2 cells. ( A ) PCR results of LMP2 and GLuc amplification using the TC-1-GLUC-LMP2 cells genomic DNA as a template. M: DNA ladder; 1: TC-1-GLUC-LMP2 cells as template using primers for LMP2 ; 2: DNA of pVR-LMP2 plasmid; 3: genomic DNA of TC-1 cells; 4: TC-1-GLUC-LMP2 cells as template using primers for GLuc; 5: DNA of pCMV-Gaussia Luc; 6: genomic DNA of TC-1 cells; ( B ) RT-PCR results to verify GLuc and LMP2 mRNA expression. M: RNA ladder; 1: RT-PCR result using TC-1-GLUC-LMP2 as template amplifying LMP2 ; 2: 293 cells transfected with pVR-LMP2 as template amplifying LMP2 ; 3: TC-1 cells as template amplifying LMP2; 4: RT-PCR result using TC-1-GLUC-LMP2 as template amplifying GLuc ; 5: 293 cells transfected with pCMV-Gaussia Luc as template amplifying GLuc ; 6: TC-1 cells as template amplifying GLuc ; ( C ) Western blot results of LMP2 protein expression in TC-1-GLUC-LMP2 cells at different passages. M: PageRuler Prestained Protein Ladder; 1: Passage 1 TC-1-GLUC-LMP2 cells, LMP2 with a molecular weight of 55 KD; 2: Passage 10 TC-1-GLUC-LMP2 cells; 3: Passage 30 TC-1-GLUC-LMP2 cells; 4: TC-1 cells; ( D ) Identifying the stable expression of GLuc . The mean relative light unit (RLU) value of 10 4 TC-1-GLUC-LMP2 cells at passages 1, 10 and 30 were 7.44 × 10 5 , 7.63 × 10 5 and 7.34 × 10 5 . NS, no significant difference; each column represents mean ± SD ( n = 5) in ( D ).

Article Snippet: Membranes were incubated with EBV LMP2 monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA, #sc-101314) diluted 1:500 in 5% skimmed milk, and a horseradish peroxidase-conjugated goat anti-rat IgG (ZSGB-BIO, Beijing, China, #ZB-2307) diluted 1:1000 was used as the secondary antibody.

Techniques: Amplification, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection, Western Blot, Molecular Weight

Journal: Viruses

Article Title: Constructing TC-1-GLUC-LMP2 Model Tumor Cells to Evaluate the Anti-Tumor Effects of LMP2-Related Vaccines

doi: 10.3390/v10040145

Figure Lengend Snippet: Flow cytometry detection results. ( A ) The proportions of fluorescein isothiocyanate (FITC)-labeled cells in TC-1 was 0.2%; ( B ) The proportions of FITC-labeled cells in TC-1-GLUC-LMP2 was 98.7%. FITC was fluorescein isothiocyanate. The red area represented the detected cells.

Article Snippet: Membranes were incubated with EBV LMP2 monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA, #sc-101314) diluted 1:500 in 5% skimmed milk, and a horseradish peroxidase-conjugated goat anti-rat IgG (ZSGB-BIO, Beijing, China, #ZB-2307) diluted 1:1000 was used as the secondary antibody.

Techniques: Flow Cytometry, Labeling

Journal: Viruses

Article Title: Constructing TC-1-GLUC-LMP2 Model Tumor Cells to Evaluate the Anti-Tumor Effects of LMP2-Related Vaccines

doi: 10.3390/v10040145

Figure Lengend Snippet: Comparing the TC-1-GLUC-LMP2 and TC-1 growth curves. TC-1-GLUC-LMP2 and TC-1 cells (5 × 10 3 ) were added to E-plate L 8 plates, and cell index (CI) was measured by iCELLigence. After 120 h, both cell lines grew to 2.1 × 10 5 /cm 2 . The growth rate and adhesion were not significantly different.

Article Snippet: Membranes were incubated with EBV LMP2 monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA, #sc-101314) diluted 1:500 in 5% skimmed milk, and a horseradish peroxidase-conjugated goat anti-rat IgG (ZSGB-BIO, Beijing, China, #ZB-2307) diluted 1:1000 was used as the secondary antibody.

Techniques:

Journal: Viruses

Article Title: Constructing TC-1-GLUC-LMP2 Model Tumor Cells to Evaluate the Anti-Tumor Effects of LMP2-Related Vaccines

doi: 10.3390/v10040145

Figure Lengend Snippet: Cytotoxicity result of TC-1-GLUC-LMP2 cells. The ten groups of mixed cells were inoculated on E-plate L8 plates and incubated for 100 h. Line 1: 5 × 10 3 TC-1-GLUC-LMP2 target cells; Line 2: 5 × 10 3 TC-1-GLUC-LMP2 target cells mixed with 8 × 10 5 spleens without peptide stimulation isolated from mice immunized with vaccine-LMP2; Line 3: 5 × 10 3 target cells mixed with 8 × 10 5 spleens were spiked with LMP2-specific peptide isolated from mice immunized with vaccine-NULL; Line 4: 5 × 10 3 TC-1-GLUC-LMP2 target cells mixed with 5 × 10 4 spleens with peptide stimulation isolated from mice immunized with vaccine-LMP2; Line 5: 5 × 10 3 target cells mixed with 1 × 10 5 spleens with peptide stimulation isolated from mice immunized with vaccine-LMP2; Line 6: 5 × 10 3 target cells mixed with 2 × 10 5 spleens with peptide stimulation isolated from mice immunized with vaccine-LMP2; Line 7: 5 × 10 3 target cells mixed with 4 × 10 5 spleens with peptide stimulation isolated from mice immunized with vaccine-LMP2; Line 8: 5 × 10 3 target cells mixed with 8 × 10 5 spleens with peptide stimulation isolated from mice immunized with vaccine-LMP2; Line 9: Roswell Park Memorial Institute 1640 (RPMI 1640); Line 10: 8 × 10 5 LMP2-specific mouse spleen lymphocytes.

Article Snippet: Membranes were incubated with EBV LMP2 monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA, #sc-101314) diluted 1:500 in 5% skimmed milk, and a horseradish peroxidase-conjugated goat anti-rat IgG (ZSGB-BIO, Beijing, China, #ZB-2307) diluted 1:1000 was used as the secondary antibody.

Techniques: Incubation, Isolation

Journal: Viruses

Article Title: Constructing TC-1-GLUC-LMP2 Model Tumor Cells to Evaluate the Anti-Tumor Effects of LMP2-Related Vaccines

doi: 10.3390/v10040145

Figure Lengend Snippet: Detecting subcutaneously injected TC-1-GLUC-LMP2 cells. ( A ) The tumor in vivo was observed by the in vivo imaging system (IVIS) at 3 days post-inoculation; ( B ) The tumor was observed by the IVIS at 7 days post-inoculation; ( C ) The tumor was observed by the IVIS at 14 days post-inoculation; ( D ) IVIS results showed that the mean number of photons in the mice were 2.43 × 10 4 , 9.67 × 10 4 and 7.09 × 10 5 at three, seven, and 14 days post-inoculation, respectively; ( E ) The tumor sizes and weights of mice were recorded at 14 days after inoculation with the tumor cells; the average tumor weight and volume of the tumor-bearing mice were 0.41 g and 220 mm 3 , respectively; ( F ) Immunohistochemistry results for TC-1-GLUC-LMP2 tumors showed nuclei (blue) and cytoplasm (reddish brown), indicating that EBV LMP2 was well expressed; ( G ) Immunohistochemistry results for normal muscle tissue of mice; ( H ) HE staining results of TC-1-GLUC-LMP2 tumors showed that nuclei became larger (blue), while the cytoplasm shrank or even disappeared (pink); ( I ) HE staining results of normal muscle tissue of mice. # means the number of each mouse. *** p < 0.005; **** p < 0.001 in ( D ) ( n = 7).

Article Snippet: Membranes were incubated with EBV LMP2 monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA, #sc-101314) diluted 1:500 in 5% skimmed milk, and a horseradish peroxidase-conjugated goat anti-rat IgG (ZSGB-BIO, Beijing, China, #ZB-2307) diluted 1:1000 was used as the secondary antibody.

Techniques: Injection, In Vivo, In Vivo Imaging, Immunohistochemistry, Staining

Journal: Viruses

Article Title: Constructing TC-1-GLUC-LMP2 Model Tumor Cells to Evaluate the Anti-Tumor Effects of LMP2-Related Vaccines

doi: 10.3390/v10040145

Figure Lengend Snippet: TC-1-GLUC-LMP2 tumor challenge with LMP2-associated vaccine in vivo. ( A ) The number of tumor photons in the vaccine-NULL or vaccine-LMP2-immunized mice were detected at three, seven, 14 days with the in vivo imaging system (IVIS); ( B ) The LMP2-specific immune response was analyzed by ELISPOT after TC-1-GLUC-LMP2 tumor cells inoculation 14 days. *** p < 0.005; **** p < 0.001; NS, no significant difference; each column represents mean ± SD ( n = 5).

Article Snippet: Membranes were incubated with EBV LMP2 monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA, #sc-101314) diluted 1:500 in 5% skimmed milk, and a horseradish peroxidase-conjugated goat anti-rat IgG (ZSGB-BIO, Beijing, China, #ZB-2307) diluted 1:1000 was used as the secondary antibody.

Techniques: In Vivo, In Vivo Imaging, Enzyme-linked Immunospot