Bone marrow cells were sterilely harvested from tibiae and femurs of C3H/HeJ mice (stock#000659, The Jackson laboratory, Bar Harbor, Maine; Resistance to lipopolysaccharide or LPS due to a spontaneous mutation in toll-like receptor 4 or tlr4 gene). After lysing red blood cells, bone marrow cells were cultured at the cell density of 2 × 106/ml in RPMI 1640medium in 10cm dish (BD Biosciences, Durham, NC) at 37 °C, 5% CO2, supplemented with 10% fetal calf serum (FCS, cat# 100-106, Gemini Bio-Products, West Sacramento, CA), 2 mM L-glutamine (cat#G7513, Sigma, St. Louis, MO), 1X MEM non-essential amino acids (cat# 11140-050, Gibco, Grand island, NY), 1mM sodium pyruvate (cat#25-000-CI, Cellgro, Manassas, VA), 0.5mM beta-mercaptoethanol (cat#M3148, Sigma),100U/ml penicillin G, 100μg/ml streptomycin (cat#30-002-CI, Cellgro). For making neutrophils, the bone marrow cells were cultured in the growth medium that contains 100ng/ml recombinant G-CSF (cat# 250-05, PeproTech) alone. Every 3 days, the floating cells were harvested and re-cultured in fresh growth medium. On day 8, cells floating in the supernatants were subjected to flow cytometry analysis for Ly-6G or Gr1 positivity. When the positivity reached >80%, the cells were used as neutrophils in individual experiments.
Recombinant g csf
Manufactured by Thermo Fisher Scientific
Sourced in United States
Recombinant G-CSF is a laboratory reagent produced using recombinant DNA technology. It is a granulocyte colony-stimulating factor that stimulates the production and differentiation of neutrophilic granulocytes and stem cells.
Automatically generated - may contain errors
Lab products found in correlation
Sodium pyruvate, by Corning (2 mentions)
Penicillin g, by Corning (2 mentions)
Mem non essential amino acid, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Merck Group (2 mentions)
Streptomycin, by Corning (2 mentions)
β mercaptoethanol, by Merck Group (2 mentions)
C3h hej mice, by Jackson ImmunoResearch (1 mentions)
Anti ly6g iggs clone 1a8, by BioXCell (1 mentions)
Recombinant gm csf, by Thermo Fisher Scientific (1 mentions)
Ly2228820 p38 inhibitor, by Selleck Chemicals (1 mentions)
Anti ly6g biotin, by Miltenyi Biotec (1 mentions)
Cd34 microbead kit, by Miltenyi Biotec (1 mentions)
Gm csf, by Thermo Fisher Scientific (1 mentions)
Streptavidin beads, by Miltenyi Biotec (1 mentions)
Lineage depletion kit, by Miltenyi Biotec (1 mentions)
Recombinant flt3l, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Lonza (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
Aria 2 system, by BD (1 mentions)
7 protocols using recombinant g csf
1
Neutrophil Differentiation from Murine Bone Marrow
2
Immunomodulation of Tumor Microenvironment
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Mice were subcutaneously injected with 2 μg recombinant GCSF (PeproTech) in 100 μl PBS every day for 10 days. Mice bearing 1.0 cm diameter PyMT-B6 tumors were treated with 30 μg Flt3L (Celldex) daily for 9 days by intraperitoneal injection. Mice bearing 1.0 cm diameter PyMT-B6 tumors were treated with 50 μg anti-GCSF IgGs (clone 67604, R&D Systems), 500 μg anti-IL-6 IgGs (clone MP5-20F3, BioXCell), or matched isotype control three times per week by intraperitoneal injection for 2 weeks. Mice were treated with anti-Ly6G IgGs (clone 1A8, BioXCell) or matched isotype control, first dose 400 μg, 100 μg for subsequent doses, three times per week beginning at time point indicated in the experiment. Mice were treated with 200 μg anti-PD1 IgGs (clone RMP1-14, BioXCell) every 3 days by intraperitoneal injection. Mice were treated with 50 μg poly I:C every 5 days by intratumoral injection, according to time points indicated in the experiment.
3
Hematopoietic Progenitor Cell Isolation and Characterization
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Mouse. Hematopoietic progenitor cells (HPCs) were isolated from mouse bone marrow by using a Lineage depletion kit (Miltenyi), according to the manufacturer’s instructions. Cells were seeded at 25,000 cell/ml in 24-well plates and recombinant GM-CSF (20 ng/ml; Invitrogen), 20% v/v TES were added on day 1 and day 3. At day 5, Ly6G positive neutrophils were isolated by using anti-Ly6G biotin (Miltenyi) and streptavidin beads (Miltenyi), according to manufacturer’s followed by suppression assay. In addition, total cells were stained and analyzed for flow cytometry. In other experiments, LY2228820 p38 inhibitor (1 μM; Selleckchem) was added to HPC culture on day 3.
Human. Hematopoietic progenitor cells (HPCs) were isolated from cord blood using the CD34 MicroBead Kit (Miltenyi), according to the manufacturer’s instructions. Cells were seeded at 5 × 104 cell/ml in 6-well plates with recombinant G-CSF (100 ng/ml; PeproTech) and GM-CSF (10 ng/ml; PeproTech). At day 7, 30% v/v TES were added and the next day flow cytometry analysis was performed.
Human. Hematopoietic progenitor cells (HPCs) were isolated from cord blood using the CD34 MicroBead Kit (Miltenyi), according to the manufacturer’s instructions. Cells were seeded at 5 × 104 cell/ml in 6-well plates with recombinant G-CSF (100 ng/ml; PeproTech) and GM-CSF (10 ng/ml; PeproTech). At day 7, 30% v/v TES were added and the next day flow cytometry analysis was performed.
4
Intranasal IL-33 and G-CSF modulate airway inflammation
5
Isolation and differentiation of BM progenitors
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BM cells were isolated, lineage depleted, and stained as described above. CD45.2+ MPs (Lin-cKit+ScaI−), MDPs, or CDPs were isolated from whole BM of tumor-bearing or tumor-free mice by cell sorting on the ARIAII system (BD). A total of 2500 sorted progenitors were plated on 1.125 × 106 CD45.1+ BM cell feeder culture in RPMI-1640 medium (Lonza) supplemented with 10% fetal bovine serum (Atlanta Biological), β-mercaptoethanol (Gibco), non-essential amino acids (Life Technologies), and l -glutamine (Life Technologies) in the presence of 100 ng/ml recombinant Flt3L and/or 100 ng/ml recombinant GCSF (PeproTech) in 24-well plates. The medium was replaced after 3 days and cultures were analyzed after 5 days. To lift cells, 0.05% Trypsin (HyClone) was used. Cells were stained and analyzed as described for flow cytometry, identifying the progeny of isolated progenitors by CD45.2 positivity. For GCSF priming experiment, progenitors were treated with 100 ng/ml GCSF or media alone for 24 h prior to plating in differentiation assay described above.
6
Isolation and Differentiation of Murine Neutrophils
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Bone marrow cells were sterilely harvested from tibiae and femurs of C3H/HeJ mice. After lysing red blood cells, bone marrow cells were cultured at a cell density of 2×106 per ml in 10-cm dishes (BD Biosciences, Durham, NC, USA) at 37°C in an atmosphere of 5% CO2. Cells were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (cat#100-106, Gemini Bio-Products, West Sacramento, CA, USA), 2 mM l-glutamine (cat#G7513, Sigma, St. Louis, MO, USA), 1X MEM non-essential amino acids (cat#11140-050, Gibco, Grand Island, NY, USA), 1 mM sodium pyruvate (cat#25-000-CI, Cellgro, Manassas, VA, USA), 0.5 mM beta-mercaptoethanol (cat#M3148, Sigma), 100 U/ml penicillin G, and 100 μg/ml streptomycin (cat#30-002-CI, Cellgro).
To make neutrophils, bone marrow cells were cultured in growth medium containing 100 ng/ml recombinant G-CSF (cat#250-05, PeproTech). Every 3 days, floating cells were harvested and re-cultured in fresh growth medium. On day 8, cells floating in the supernatants were analyzed by flow cytometry for the presence of Ly-6G or Gr1. When positivity was >80%, the cells were used as neutrophils in chemotaxis assays.
To make neutrophils, bone marrow cells were cultured in growth medium containing 100 ng/ml recombinant G-CSF (cat#250-05, PeproTech). Every 3 days, floating cells were harvested and re-cultured in fresh growth medium. On day 8, cells floating in the supernatants were analyzed by flow cytometry for the presence of Ly-6G or Gr1. When positivity was >80%, the cells were used as neutrophils in chemotaxis assays.
7
Neutrophil Transfection and Culture
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Splenic neutrophils were sorted by the Anti-Ly-6G MicroBeads UltraPure kit (Catalog# 130-120-337, Miltenyi Biotec). Transient transfection of neutrophils was done as previously described.80 (link) In brief, three million neutrophils were electroporated with 300 nM of SMARTpool ON-TARGETplus siRNA which combines four gene-specific siRNAs into a single reagent pool using the P3 Primary Cell 4D-Nucleofector X Kit (Lonza, Switzerland) under human monocyte nucleofection program with an Amaxa electroporation system. The cells were then cultured for 48 h in the medium supplied with the kit containing 10% FBS and 30 ng/mL recombinant G-CSF (PeproTech, Rocky Hill, NJ).
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