Journal: International journal of molecular sciences

Article Title: NIH/3T3 Fibroblasts Selectively Activate T Cells Specific for Posttranslationally Modified Collagen Type II.

doi: 10.3390/ijms241310811

Figure Lengend Snippet: Figure 1. Expression of the Aq molecule on NIH/3T3 cells (A), thymic fibroblasts (B) and LS48 cells (C). After adhesion for 24 h, the cells were stained with biotin labeled anti-mouse Aq (clone KH116) and FITC Streptavidin for 15 min at 37 ◦C in dark. Photomicrographs show light/fluorescence from the respective area. Bar 100 µm.

Article Snippet: NIH/3T3 Cell Line NIH/3T3 (American Type Culture Collection, Rockville, MD, USA, ATCC® CRL1658TM) is a continuous fibroblast cell line derived from NIH/Swiss mouse embryo cultures by the same method as the original random-bred 3T3 (ATCC® CCL-92TM) and the inbred BALB/c 3T3 (ATCC® CCL-163TM) [64].

Techniques: Expressing, Staining, Labeling

Journal: International journal of molecular sciences

Article Title: NIH/3T3 Fibroblasts Selectively Activate T Cells Specific for Posttranslationally Modified Collagen Type II.

doi: 10.3390/ijms241310811

Figure Lengend Snippet: Figure 2. Antigen-presenting capacity of different types of fibroblasts. The ability of LS48, NIH/3T3, synovial fibroblasts (SF), thymic fibroblasts (ThyF), and dermal fibroblasts (DF) to process and present COL2 was evaluated by measuring IL-2 levels in culture medium following 24 h co-culture of fibroblasts with COL2-specific HCQ.3 T-cell hybridoma and 50 µg/mL COL2. The mean of triplicate measurements for each sample is shown.

Article Snippet: NIH/3T3 Cell Line NIH/3T3 (American Type Culture Collection, Rockville, MD, USA, ATCC® CRL1658TM) is a continuous fibroblast cell line derived from NIH/Swiss mouse embryo cultures by the same method as the original random-bred 3T3 (ATCC® CCL-92TM) and the inbred BALB/c 3T3 (ATCC® CCL-163TM) [64].

Techniques: Co-Culture Assay

Journal: International journal of molecular sciences

Article Title: NIH/3T3 Fibroblasts Selectively Activate T Cells Specific for Posttranslationally Modified Collagen Type II.

doi: 10.3390/ijms241310811

Figure Lengend Snippet: Figure 3. NIH/3T3 fibroblasts activate HCQ.3 cells even in the absence of COL2 antigen. (A) NIH/3T3 and HCQ.3 cells were co-cultured for 24 h with or without COL2 and glycosylated COL2259–273 peptide, as well as with or without addition of professional APCs. Murine thymic fibroblasts (B), LS48 fibroblasts (C), QB dermal fibroblasts (D), or QB synovial fibroblasts (E), and HCQ.3 cells were co-cultured for 24 h with or without COL2 and glycosylated COL2259–273 peptide, as well as with or without addition of professional APCs. All samples were assessed in triplicates. Data represent ± SEM.

Article Snippet: NIH/3T3 Cell Line NIH/3T3 (American Type Culture Collection, Rockville, MD, USA, ATCC® CRL1658TM) is a continuous fibroblast cell line derived from NIH/Swiss mouse embryo cultures by the same method as the original random-bred 3T3 (ATCC® CCL-92TM) and the inbred BALB/c 3T3 (ATCC® CCL-163TM) [64].

Techniques: Cell Culture

Journal: International journal of molecular sciences

Article Title: NIH/3T3 Fibroblasts Selectively Activate T Cells Specific for Posttranslationally Modified Collagen Type II.

doi: 10.3390/ijms241310811

Figure Lengend Snippet: Figure 4. Ability of NIH/3T3 fibroblasts to activate different COL2-specific T-cell hybridomas. (A) NIH/3T3 fibroblasts were co-cultured with different COL2-specific hybridomas that recognize either the non-modified COL2259–273 epitope or the posttranslationally modified (glycosylated and hydroxylated) immunodominant epitope. Upon 24 h of incubation, IL-2 levels in culture supernatant were measured by enzyme-linked immunosorbent assay (ELISA). (B) HCQ.3 hybridoma was co- cultured with different types of fibroblasts—NIH/3T3, LS48, QB DF, QB SF, and ThyF. After 24 h, hybridoma activation was evaluated by measuring IL-2 content in co-culture supernatant. (C) Co- culture of BW 5147 cells with NIH/3T3 or LS48, QB DF, QB SF, and ThyF. Following 24 h of incubation, IL-2 levels in culture supernatant were measured by ELISA. All samples were analyzed in triplicate. Data represent ± SEM.

Article Snippet: NIH/3T3 Cell Line NIH/3T3 (American Type Culture Collection, Rockville, MD, USA, ATCC® CRL1658TM) is a continuous fibroblast cell line derived from NIH/Swiss mouse embryo cultures by the same method as the original random-bred 3T3 (ATCC® CCL-92TM) and the inbred BALB/c 3T3 (ATCC® CCL-163TM) [64].

Techniques: Cell Culture, Incubation, Enzyme-linked Immunosorbent Assay, Activation Assay, Co-Culture Assay

Journal: International journal of molecular sciences

Article Title: NIH/3T3 Fibroblasts Selectively Activate T Cells Specific for Posttranslationally Modified Collagen Type II.

doi: 10.3390/ijms241310811

Figure Lengend Snippet: Figure 5. NIH/3T3 fibroblasts interaction with COL2-specific T-cell hybridomas is cell-cell contact- dependent. NIH/3T3 fibroblasts and COL2-specific T-cell hybridomas were co-cultured for 24 h with or without transwell membrane. After 24 h of incubation, IL-2 levels in culture supernatant were measured by ELISA. Data represent ± SEM. All samples were assayed in triplicate.

Article Snippet: NIH/3T3 Cell Line NIH/3T3 (American Type Culture Collection, Rockville, MD, USA, ATCC® CRL1658TM) is a continuous fibroblast cell line derived from NIH/Swiss mouse embryo cultures by the same method as the original random-bred 3T3 (ATCC® CCL-92TM) and the inbred BALB/c 3T3 (ATCC® CCL-163TM) [64].

Techniques: Cell Culture, Membrane, Incubation, Enzyme-linked Immunosorbent Assay

Journal: International journal of molecular sciences

Article Title: NIH/3T3 Fibroblasts Selectively Activate T Cells Specific for Posttranslationally Modified Collagen Type II.

doi: 10.3390/ijms241310811

Figure Lengend Snippet: Figure 6. Expression levels of MHCII and CD40L on different types of murine fibroblasts. NIH/3T3, LS48, and QB synovial fibroblasts were cultured in standard complete DMEM medium or in medium with 100 U/mL IFN-γ for 72 h. After that, the cells were trypsinized, stained with fluorochrome- labeled antibodies, and analyzed by flow-cytometry. (A) Untreated and IFN-γ-pulsed NIH/3T3, LS48, and QB synovial fibroblasts were analyzed for MHC class II molecules expression using monoclonal MHCII antibody (clone M5/114). (B) Expression levels of CD40L on different types of murine fibroblasts following standard culture and INF-γ treatment. (C) CD40, CD40L, and MHCII on NIH/3T3 cells were blocked using specific monoclonal antibodies. The fibroblasts were then washed and co-cultured with HCQ.3 T-cell hybridomas. After 24 h of incubation, the IL-2 levels in the culture medium were analyzed by ELISA.

Article Snippet: NIH/3T3 Cell Line NIH/3T3 (American Type Culture Collection, Rockville, MD, USA, ATCC® CRL1658TM) is a continuous fibroblast cell line derived from NIH/Swiss mouse embryo cultures by the same method as the original random-bred 3T3 (ATCC® CCL-92TM) and the inbred BALB/c 3T3 (ATCC® CCL-163TM) [64].

Techniques: Expressing, Cell Culture, Staining, Labeling, Cytometry, Bioprocessing, Incubation, Enzyme-linked Immunosorbent Assay

Journal: International journal of molecular sciences

Article Title: NIH/3T3 Fibroblasts Selectively Activate T Cells Specific for Posttranslationally Modified Collagen Type II.

doi: 10.3390/ijms241310811

Figure Lengend Snippet: Figure 7. Effect of NIH/3T3 cell surface molecules blocking HCQ.3 activation. (A) NIH/3T3 cells were incubated for 1 h with anti-mouse CD106 or CD34 antibodies. HCQ.3 cells were treated with anti-mouse CD33, CD4, or TCR Vβ8 antibodies for 1 h. Thereafter, the cells were washed and co-cultured with untreated HCQ.3 or NIH/3T3 cells. After 24 h of co-culture, T-cell hybridoma activation was analyzed based on IL-2 measurements in culture medium. (B) NIH/3T3 fibroblasts were treated with anti-mouse CD44, CD55, CD90.2, or CD138 antibodies for 1 h. After that, the cells were washed and co-cultured with HCQ.3 T-cell hybridomas for 24 h. IL-2 content was measured by ELISA following co-culture. All samples were plated and analyzed in triplicate. Data are shown as ± SEM. * p < 0.05, ** p < 0.01 vs. NIH/3T3 + HCQ.3.

Article Snippet: NIH/3T3 Cell Line NIH/3T3 (American Type Culture Collection, Rockville, MD, USA, ATCC® CRL1658TM) is a continuous fibroblast cell line derived from NIH/Swiss mouse embryo cultures by the same method as the original random-bred 3T3 (ATCC® CCL-92TM) and the inbred BALB/c 3T3 (ATCC® CCL-163TM) [64].

Techniques: Blocking Assay, Activation Assay, Incubation, Cell Culture, Co-Culture Assay, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: NIH/3T3 Fibroblasts Selectively Activate T Cells Specific for Posttranslationally Modified Collagen Type II

doi: 10.3390/ijms241310811

Figure Lengend Snippet: Expression of the A q molecule on NIH/3T3 cells ( A ), thymic fibroblasts ( B ) and LS48 cells ( C ). After adhesion for 24 h, the cells were stained with biotin labeled anti-mouse A q (clone KH116) and FITC Streptavidin for 15 min at 37 °C in dark. Photomicrographs show light/fluorescence from the respective area. Bar 100 µm.

Article Snippet: NIH/3T3 (American Type Culture Collection, Rockville, MD, USA, ATCC ® CRL-1658™) is a continuous fibroblast cell line derived from NIH/Swiss mouse embryo cultures by the same method as the original random-bred 3T3 (ATCC ® CCL-92™) and the inbred BALB/c 3T3 (ATCC ® CCL-163™) [ ].

Techniques: Expressing, Staining, Labeling, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: NIH/3T3 Fibroblasts Selectively Activate T Cells Specific for Posttranslationally Modified Collagen Type II

doi: 10.3390/ijms241310811

Figure Lengend Snippet: Antigen-presenting capacity of different types of fibroblasts. The ability of LS48, NIH/3T3, synovial fibroblasts (SF), thymic fibroblasts (ThyF), and dermal fibroblasts (DF) to process and present COL2 was evaluated by measuring IL-2 levels in culture medium following 24 h co-culture of fibroblasts with COL2-specific HCQ.3 T-cell hybridoma and 50 µg/mL COL2. The mean of triplicate measurements for each sample is shown.

Article Snippet: NIH/3T3 (American Type Culture Collection, Rockville, MD, USA, ATCC ® CRL-1658™) is a continuous fibroblast cell line derived from NIH/Swiss mouse embryo cultures by the same method as the original random-bred 3T3 (ATCC ® CCL-92™) and the inbred BALB/c 3T3 (ATCC ® CCL-163™) [ ].

Techniques: Co-Culture Assay

Journal: International Journal of Molecular Sciences

Article Title: NIH/3T3 Fibroblasts Selectively Activate T Cells Specific for Posttranslationally Modified Collagen Type II

doi: 10.3390/ijms241310811

Figure Lengend Snippet: NIH/3T3 fibroblasts activate HCQ.3 cells even in the absence of COL2 antigen. ( A ) NIH/3T3 and HCQ.3 cells were co-cultured for 24 h with or without COL2 and glycosylated COL2 259–273 peptide, as well as with or without addition of professional APCs. Murine thymic fibroblasts ( B ), LS48 fibroblasts ( C ), QB dermal fibroblasts ( D ), or QB synovial fibroblasts ( E ), and HCQ.3 cells were co-cultured for 24 h with or without COL2 and glycosylated COL2 259–273 peptide, as well as with or without addition of professional APCs. All samples were assessed in triplicates. Data represent ± SEM.

Article Snippet: NIH/3T3 (American Type Culture Collection, Rockville, MD, USA, ATCC ® CRL-1658™) is a continuous fibroblast cell line derived from NIH/Swiss mouse embryo cultures by the same method as the original random-bred 3T3 (ATCC ® CCL-92™) and the inbred BALB/c 3T3 (ATCC ® CCL-163™) [ ].

Techniques: Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: NIH/3T3 Fibroblasts Selectively Activate T Cells Specific for Posttranslationally Modified Collagen Type II

doi: 10.3390/ijms241310811

Figure Lengend Snippet: Ability of NIH/3T3 fibroblasts to activate different COL2-specific T-cell hybridomas. ( A ) NIH/3T3 fibroblasts were co-cultured with different COL2-specific hybridomas that recognize either the non-modified COL2 259–273 epitope or the posttranslationally modified (glycosylated and hydroxylated) immunodominant epitope. Upon 24 h of incubation, IL-2 levels in culture supernatant were measured by enzyme-linked immunosorbent assay (ELISA). ( B ) HCQ.3 hybridoma was co-cultured with different types of fibroblasts—NIH/3T3, LS48, QB DF, QB SF, and ThyF. After 24 h, hybridoma activation was evaluated by measuring IL-2 content in co-culture supernatant. ( C ) Co-culture of BW 5147 cells with NIH/3T3 or LS48, QB DF, QB SF, and ThyF. Following 24 h of incubation, IL-2 levels in culture supernatant were measured by ELISA. All samples were analyzed in triplicate. Data represent ± SEM.

Article Snippet: NIH/3T3 (American Type Culture Collection, Rockville, MD, USA, ATCC ® CRL-1658™) is a continuous fibroblast cell line derived from NIH/Swiss mouse embryo cultures by the same method as the original random-bred 3T3 (ATCC ® CCL-92™) and the inbred BALB/c 3T3 (ATCC ® CCL-163™) [ ].

Techniques: Cell Culture, Modification, Incubation, Enzyme-linked Immunosorbent Assay, Activation Assay, Co-Culture Assay

Journal: International Journal of Molecular Sciences

Article Title: NIH/3T3 Fibroblasts Selectively Activate T Cells Specific for Posttranslationally Modified Collagen Type II

doi: 10.3390/ijms241310811

Figure Lengend Snippet: NIH/3T3 fibroblasts interaction with COL2-specific T-cell hybridomas is cell-cell contact-dependent. NIH/3T3 fibroblasts and COL2-specific T-cell hybridomas were co-cultured for 24 h with or without transwell membrane. After 24 h of incubation, IL-2 levels in culture supernatant were measured by ELISA. Data represent ± SEM. All samples were assayed in triplicate.

Article Snippet: NIH/3T3 (American Type Culture Collection, Rockville, MD, USA, ATCC ® CRL-1658™) is a continuous fibroblast cell line derived from NIH/Swiss mouse embryo cultures by the same method as the original random-bred 3T3 (ATCC ® CCL-92™) and the inbred BALB/c 3T3 (ATCC ® CCL-163™) [ ].

Techniques: Cell Culture, Membrane, Incubation, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: NIH/3T3 Fibroblasts Selectively Activate T Cells Specific for Posttranslationally Modified Collagen Type II

doi: 10.3390/ijms241310811

Figure Lengend Snippet: Expression levels of MHCII and CD40L on different types of murine fibroblasts. NIH/3T3, LS48, and QB synovial fibroblasts were cultured in standard complete DMEM medium or in medium with 100 U/mL IFN-γ for 72 h. After that, the cells were trypsinized, stained with fluorochrome-labeled antibodies, and analyzed by flow-cytometry. ( A ) Untreated and IFN-γ-pulsed NIH/3T3, LS48, and QB synovial fibroblasts were analyzed for MHC class II molecules expression using monoclonal MHCII antibody (clone M5/114). ( B ) Expression levels of CD40L on different types of murine fibroblasts following standard culture and INF-γ treatment. ( C ) CD40, CD40L, and MHCII on NIH/3T3 cells were blocked using specific monoclonal antibodies. The fibroblasts were then washed and co-cultured with HCQ.3 T-cell hybridomas. After 24 h of incubation, the IL-2 levels in the culture medium were analyzed by ELISA.

Article Snippet: NIH/3T3 (American Type Culture Collection, Rockville, MD, USA, ATCC ® CRL-1658™) is a continuous fibroblast cell line derived from NIH/Swiss mouse embryo cultures by the same method as the original random-bred 3T3 (ATCC ® CCL-92™) and the inbred BALB/c 3T3 (ATCC ® CCL-163™) [ ].

Techniques: Expressing, Cell Culture, Staining, Labeling, Flow Cytometry, Incubation, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: NIH/3T3 Fibroblasts Selectively Activate T Cells Specific for Posttranslationally Modified Collagen Type II

doi: 10.3390/ijms241310811

Figure Lengend Snippet: Effect of NIH/3T3 cell surface molecules blocking HCQ.3 activation. ( A ) NIH/3T3 cells were incubated for 1 h with anti-mouse CD106 or CD34 antibodies. HCQ.3 cells were treated with anti-mouse CD33, CD4, or TCR Vβ8 antibodies for 1 h. Thereafter, the cells were washed and co-cultured with untreated HCQ.3 or NIH/3T3 cells. After 24 h of co-culture, T-cell hybridoma activation was analyzed based on IL-2 measurements in culture medium. ( B ) NIH/3T3 fibroblasts were treated with anti-mouse CD44, CD55, CD90.2, or CD138 antibodies for 1 h. After that, the cells were washed and co-cultured with HCQ.3 T-cell hybridomas for 24 h. IL-2 content was measured by ELISA following co-culture. All samples were plated and analyzed in triplicate. Data are shown as ± SEM. * p ˂ 0.05, ** p ˂ 0.01 vs. NIH/3T3 + HCQ.3.

Article Snippet: NIH/3T3 (American Type Culture Collection, Rockville, MD, USA, ATCC ® CRL-1658™) is a continuous fibroblast cell line derived from NIH/Swiss mouse embryo cultures by the same method as the original random-bred 3T3 (ATCC ® CCL-92™) and the inbred BALB/c 3T3 (ATCC ® CCL-163™) [ ].

Techniques: Blocking Assay, Activation Assay, Incubation, Cell Culture, Co-Culture Assay, Enzyme-linked Immunosorbent Assay