mouse op9 cell line gfp expressing  (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