Nucleated cells from the femur and tibia of 3-week post-pIpC mice were lineage depleted with a MACS lineage cell separation kit according to manufacturer’s instructions (Miltenyi Biotec, Auburn, CA). Lineage depleted cells were cultured onto 30,000 OP9 cells (plated night before) in IMDM supplemented with 10% defined FBS, 55mM BME, 50 U/ml penicillin, 50mg/mL streptomycin, 0.1mM Glutamax, 10ng/mL Flt3L, and 5ng/mL IL-7. Recombinant mouse cytokines were obtained from R&D Systems (Minneapolis, MN) or Invitrogen (Carlsbad, CA). Cells were transferred onto fresh OP9 cells every 3 days. To evaluate myeloid and B cell differentiation, cells were analyzed 6 days after culture with B220-APC and CD11b-APC/cy7 antibodies (BioLegend, San Diego, CA).
B220 apc and cd11b apc cy7 antibodies
Manufactured by BioLegend
B220-APC is a mouse monoclonal antibody that binds to the B220 (CD45R) antigen, a pan-B cell marker. CD11b-APC/cy7 is a mouse monoclonal antibody that binds to the CD11b antigen, which is expressed on monocytes, macrophages, and granulocytes.
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3 protocols using b220 apc and cd11b apc cy7 antibodies
1
Lineage Depletion and Cell Culture Protocol
2
Myeloid and B-cell Differentiation Assay
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Nucleated cells from the femur and tibia of 5–6 week old mice were lineage depleted with a MACS lineage cell separation kit according to manufacturer’s instructions (Miltenyi Biotec, Auburn, CA). Lineage depleted cells were cultured onto 30,000 OP9 cells (plated night before) in IMDM supplemented with 10% defined FBS, 55mM BME, 50 U/ml penicillin, 50mg/mL streptomycin, 0.1mM Glutamax, 5ng/mL Flt3L, and 1 ng/mL IL-7. Cultures were treated with DMSO control, 1 uM of BMP inhibitor LDN 193189 (Abcam, Cambridge, MA), or 100nM FoxO1 inhibitor AS1842856 (Calbiochem, Billerica, MA). Mouse cytokines were obtained from R&D Systems or Invitrogen. Cells were transferred onto fresh OP9 cells every 3 days. To evaluate myeloid and B-cell differentiation in cultures, cells were analyzed 6 or 9 days after culture with B220-APC and CD11b-APC/cy7 antibodies (BioLegend, San Diego, CA) by flow cytometry on the Beckman Coulter FC500 flow cytometer.
3
Hematopoietic Lineage Commitment Assay
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Nucleated cells from the femur and tibia of 5-to 6-wk-old mice were lineage depleted with a MACS Lineage Cell Separation Kit, according to the manufacturer's instructions (Miltenyi Biotec). Lin 2 cells were cultured onto 40,000 OP9 cells (plated the night before) in IMDM, supplemented with 10% defined FBS, 55 mM 2-ME, 50 U/ml penicillin, 50 mg/ml streptomycin, 0.1 mM GlutaMAX, 5 ng/ml Flt3L, and 1 ng/ml IL-7. EML cells overexpressing MSCV, or MSCV-mirn23a were cultured onto 40,000 OP9 cells (plated the night before) in IMDM, supplemented with 20% FBS, 10% COS-KSL conditioned media (source of SCF), 5 ng/ml Flt3L, and 1 ng/ml IL-7. Recombinant mouse cytokines were obtained from R&D Systems or Thermo Fisher Scientific Life Sciences. Cells were transferred onto fresh OP9 cells every 3 d. To evaluate myeloid and B cell differentiation in nontransduced cultures, cells were analyzed 6 d after culture with B220-APC and CD11b-APC/ Cy7 antibodies (BioLegend). In MSCV or MSCV-Trib3-overexpressing hematopoietic cultures, cells were analyzed 9 d after OP9 culture.
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