mouse op9 cell line  (ATCC)

Journal: European journal of immunology

Article Title: Guidelines for mouse and human DC generation

doi: 10.1002/eji.202249816

Figure Lengend Snippet: Selected protocols for in vitro simultaneous generation of human cDC1, cDC2, pDC, and eventually DC3 from CD34 + HSC

Article Snippet: MEM Alpha Medium w/o Nucleosides (αMEM) (Life Technologies, Cat no 22561–021) Fetal Bovine/Calf Serum (FBS/FCS; test and use the same batch throughout), filtered and heat-inactivated Mouse OP9 cell line (GFP-expressing) (ATCC, Cat no CRL-2749) Dulbecco’s Phosphate Buffered Saline (DPBS; Sigma, Cat no D8537) Penicillin-Streptomycin (Sigma, Cat no P0781) Ethylenediaminetetraacetic acid (EDTA, Sigma, Cat no E7889) Recombinant human cytokines: Flt-3 ligand (Flt3L; Immunotools, Cat no 11343305); Stem Cell Factor (SCF; Immunotools, Cat no 11343325); GM-CSF (R&D systems, Cat no CAA26822) OP9 medium: αMEM, 20% FCS, penicillin-streptomycin Trypsin-EDTA solution: 0.25% Trypsin, 0.53mM EDTA DC differentiation medium: αMEM, 10% FCS, 1% penicillin-streptomycin, 100ng/ml Flt3L, 20ng/ml SCF, 20mg/ml GM-CSF 96 Well TC-Treated Microplates (round-bottom) (Corning ® , Cat no CLS3799) 15 ml or 50 ml sterile polypropylene conical Falcon tubes (Greiner, Cat nos 188271 and 227261)

Techniques: In Vitro, Generated, Functional Assay, Expressing

Journal: European journal of immunology

Article Title: Guidelines for mouse and human DC generation

doi: 10.1002/eji.202249816

Figure Lengend Snippet: Flow cytometric analysis and DC output of CD34 + differentiation. (A) Flow cytometric analysis of the culture at Day 21, using LSR-Fortessa X20 (BD biosciences) and the panel described in . Debris, doublets, and dead cells are first excluded, followed by OP9 (GFP + in the FITC channel and CD45 − ) and CD45 low CD15 + granulocyte precursors. Selecting lineage (CD3, 16, 19, 20, 34, 56) negative HLA-DR + cells excludes lymphoid lineages and undifferentiated cells and selects antigen-presenting cells. DC subsets may then be identified by sequential gating: cDC1, CLEC9A + CD141 + ; cDC2, CD1c + CD2 + (CD163 − CD14 − ), DC3, CD1c + CD2 + CD14 + CD163 + ; Monocytes, CD11c + CD14 + (CD1c-CD2 − ); pDC, CD123 + CD303/304 + . Dimensionality reduction analyses (such as tSNE or UMAP) may also be used to visualize the discrete populations. (B) DC generation in culture at Days 3, 5, 7, 9, 11, 14, and 21, expressed as the number of subset-specific DC generated per CD34 + HSPC seeded at Day 0. Cells were identified phenotypically as shown in (A). (C) Number of DC generated from CD34 + HSPC isolated from BM or peripheral blood (PB), expressed as the number of subset-specific DC generated per CD34 + HSPC seeded at Day 0.

Article Snippet: MEM Alpha Medium w/o Nucleosides (αMEM) (Life Technologies, Cat no 22561–021) Fetal Bovine/Calf Serum (FBS/FCS; test and use the same batch throughout), filtered and heat-inactivated Mouse OP9 cell line (GFP-expressing) (ATCC, Cat no CRL-2749) Dulbecco’s Phosphate Buffered Saline (DPBS; Sigma, Cat no D8537) Penicillin-Streptomycin (Sigma, Cat no P0781) Ethylenediaminetetraacetic acid (EDTA, Sigma, Cat no E7889) Recombinant human cytokines: Flt-3 ligand (Flt3L; Immunotools, Cat no 11343305); Stem Cell Factor (SCF; Immunotools, Cat no 11343325); GM-CSF (R&D systems, Cat no CAA26822) OP9 medium: αMEM, 20% FCS, penicillin-streptomycin Trypsin-EDTA solution: 0.25% Trypsin, 0.53mM EDTA DC differentiation medium: αMEM, 10% FCS, 1% penicillin-streptomycin, 100ng/ml Flt3L, 20ng/ml SCF, 20mg/ml GM-CSF 96 Well TC-Treated Microplates (round-bottom) (Corning ® , Cat no CLS3799) 15 ml or 50 ml sterile polypropylene conical Falcon tubes (Greiner, Cat nos 188271 and 227261)

Techniques: Generated, Isolation

Journal: European journal of immunology

Article Title: Guidelines for mouse and human DC generation

doi: 10.1002/eji.202249816

Figure Lengend Snippet: Selected protocols for in vitro simultaneous generation of human cDC1, cDC2, pDC, and eventually DC3 from CD34 + HSC

Article Snippet: MEM Alpha Medium w/o Nucleosides (αMEM) (Life Technologies, Cat no 22561–021) Fetal Bovine/Calf Serum (FBS/FCS; test and use the same batch throughout), filtered and heat-inactivated Mouse OP9 cell line (GFP-expressing) (ATCC, Cat no CRL-2749) Dulbecco’s Phosphate Buffered Saline (DPBS; Sigma, Cat no D8537) Penicillin-Streptomycin (Sigma, Cat no P0781) Ethylenediaminetetraacetic acid (EDTA, Sigma, Cat no E7889) Recombinant human cytokines: Flt-3 ligand (Flt3L; Immunotools, Cat no 11343305); Stem Cell Factor (SCF; Immunotools, Cat no 11343325); GM-CSF (R&D systems, Cat no CAA26822) OP9 medium: αMEM, 20% FCS, penicillin-streptomycin Trypsin-EDTA solution: 0.25% Trypsin, 0.53mM EDTA DC differentiation medium: αMEM, 10% FCS, 1% penicillin-streptomycin, 100ng/ml Flt3L, 20ng/ml SCF, 20mg/ml GM-CSF 96 Well TC-Treated Microplates (round-bottom) (Corning ® , Cat no CLS3799) 15 ml or 50 ml sterile polypropylene conical Falcon tubes (Greiner, Cat nos 188271 and 227261)

Techniques: In Vitro, Generated, Functional Assay, Expressing

Journal: European journal of immunology

Article Title: Guidelines for mouse and human DC generation

doi: 10.1002/eji.202249816

Figure Lengend Snippet: Flow cytometric analysis and DC output of CD34 + differentiation. (A) Flow cytometric analysis of the culture at Day 21, using LSR-Fortessa X20 (BD biosciences) and the panel described in . Debris, doublets, and dead cells are first excluded, followed by OP9 (GFP + in the FITC channel and CD45 − ) and CD45 low CD15 + granulocyte precursors. Selecting lineage (CD3, 16, 19, 20, 34, 56) negative HLA-DR + cells excludes lymphoid lineages and undifferentiated cells and selects antigen-presenting cells. DC subsets may then be identified by sequential gating: cDC1, CLEC9A + CD141 + ; cDC2, CD1c + CD2 + (CD163 − CD14 − ), DC3, CD1c + CD2 + CD14 + CD163 + ; Monocytes, CD11c + CD14 + (CD1c-CD2 − ); pDC, CD123 + CD303/304 + . Dimensionality reduction analyses (such as tSNE or UMAP) may also be used to visualize the discrete populations. (B) DC generation in culture at Days 3, 5, 7, 9, 11, 14, and 21, expressed as the number of subset-specific DC generated per CD34 + HSPC seeded at Day 0. Cells were identified phenotypically as shown in (A). (C) Number of DC generated from CD34 + HSPC isolated from BM or peripheral blood (PB), expressed as the number of subset-specific DC generated per CD34 + HSPC seeded at Day 0.

Article Snippet: MEM Alpha Medium w/o Nucleosides (αMEM) (Life Technologies, Cat no 22561–021) Fetal Bovine/Calf Serum (FBS/FCS; test and use the same batch throughout), filtered and heat-inactivated Mouse OP9 cell line (GFP-expressing) (ATCC, Cat no CRL-2749) Dulbecco’s Phosphate Buffered Saline (DPBS; Sigma, Cat no D8537) Penicillin-Streptomycin (Sigma, Cat no P0781) Ethylenediaminetetraacetic acid (EDTA, Sigma, Cat no E7889) Recombinant human cytokines: Flt-3 ligand (Flt3L; Immunotools, Cat no 11343305); Stem Cell Factor (SCF; Immunotools, Cat no 11343325); GM-CSF (R&D systems, Cat no CAA26822) OP9 medium: αMEM, 20% FCS, penicillin-streptomycin Trypsin-EDTA solution: 0.25% Trypsin, 0.53mM EDTA DC differentiation medium: αMEM, 10% FCS, 1% penicillin-streptomycin, 100ng/ml Flt3L, 20ng/ml SCF, 20mg/ml GM-CSF 96 Well TC-Treated Microplates (round-bottom) (Corning ® , Cat no CLS3799) 15 ml or 50 ml sterile polypropylene conical Falcon tubes (Greiner, Cat nos 188271 and 227261)

Techniques: Generated, Isolation

Journal: Cell Death Discovery

Article Title: Pathological roles of bone marrow adipocyte-derived monocyte chemotactic protein-1 in type 2 diabetic mice

doi: 10.1038/s41420-023-01708-3

Figure Lengend Snippet: A , B The MCP-1 levels in OP9A-CM/BMA-CM were overtly higher than those in OP9-CM/BMSCs-CM, with further increases observed in HG-OP9A-CM/HG-BMA-CM compared to OP9A-CM/BMA-CM ( n = 16 and 12). C , D Compared to OP9-CM/BMSCs-CM, OP9A-CM/BMA-CM significantly inhibited the proliferation of Min6 cells at 24 h and 48 h of co-culture, and HG-OP9A-CM/HG-BMA-CM further inhibited Min6 cells proliferation ( n = 12). E , F Compared to OP9-CM/BMSCs-CM, OP9A-CM/BMA-CM significantly aggravated the apoptosis of Min6 cells, and HG-OP9A-CM/HG-BMA-CM exacerbated the apoptosis of Min6 cells ( n = 6). G GSIS in Min6 cells increased by 2-fold under Ctrl condition, 1.6-fold with OP9-CM intervention, and 1.3, 0.4 and 0.2-fold with HG-OP9-CM, OP9A-CM and HG-OP9A-CM interventions, respectively ( n = 12). H GSIS in Min6 cells increased by 2.2-fold after BMSCs-CM intervention, while HG-BMSCs-CM, BMA-CM and HG-BMA-CM interventions resulted in increases of 0.7, 0.4 and 0.09-fold, respectively ( n = 8). I The expression levels of t-Akt in Min6 cells in different intervention groups did not change dramatically compared to each other. Whereas the protein levels of p-Akt in Min6 cells were obviously decreased in the OP9A-CM/HG-OP9A-CM intervention group compared to the OP9-CM intervention group ( n = 5). J Treatment with 10, 50 and 100 ng/mL of MCP-1 resulted in a gradual decrease in p-Akt protein levels of in Min6 cells compared to the Ctrl group ( n = 5). K Compared to the Ctrl + PBS group, OP9-CM/HG-OP9-CM + 100 ng/mL Mcp-1 significantly decreased the proliferation of Min6 cells. Conversely, pretreatment of Min6 cells with the MCP-1 receptor CCR2 antagonist RS (100 µM), followed by co-culture with OP9A-CM/HG-OP9A-CM for 24 or 48 h, did not significantly alter cell viability ( n = 12). L The protein level of p-Akt in Min6 cells after OP9-CM/HG-OP9-CM + 100 ng/mL MCP-1 intervention was obviously decreased compared to the Ctrl group, while the p-Akt protein level of Min6 cells regained to a level comparable to that of the Ctrl group when RS was added to block MCP-1 pathway ( n = 5). * p < 0.05, ** p < 0.01, ns means no statistical difference.

Article Snippet: The mouse bone marrow stromal cell line OP9 was obtained from American Type Culture Collection (ATCC) and they were recently authenticated by STR profiling and tested for mycoplasma contamination.

Techniques: Co-Culture Assay, Expressing, Blocking Assay