Peripheral blood was collected using a heparinized tube from healthy adult volunteers and a patient of NHD. Peripheral blood mononuclear cells (PBMC) were isolated by Histopaque-1077 (Sigma Chemical Co., St. Louis, MO) density gradient centrifugation. PBMC were resuspended with RPMI-1640 (Nacalai Tesque, Kyoto, Japan), 10% heat-inactivated fetal bovine serum (FBS; Endotoxin = 0.692 EU/ml; Japan Bio Serum, Hiroshima, Japan) and 1% antibiotics/antimycotic (Invitrogen, Carlsbad, CA). PBMC were plated onto culture chambers at a density of 4 × 105 cells/ml and cultured overnight in standard culture conditions (37°C, 5% CO2). After overnight incubation, culture supernatant and non-adherent cells were removed. The adherent cells (monocytes) were cultured with RPMI-1640 Glutamax (Invitrogen) supplemented with 1% antibiotics/antimycotic and a mixture of the following candidate cytokines; recombinant human GM-CSF (10 ng/ml; R&D Systems, Minneapolis, MN), recombinant human IL-34 (100 ng/ml; R&D Systems) and M-CSF (10 ng/ml; Peprotec, Rocky Hill, NJ) in order to develop iMG cells. We also developed induced macrophage from human monocytes; monocytes were cultured with RPMI-1640 Glutamax supplemented with 1% antibiotics/antimycotic and recombinant human GM-CSF (10 ng/ml). All cells were cultured in standard culture conditions for up to 14 days.
Recombinant human il 34
Manufactured by R&D Systems
Sourced in United States
Recombinant human IL-34 is a cytokine that plays a role in the regulation of macrophage differentiation and function. It is produced in E. coli and purified using standard biochemical techniques.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640 glutamax, by Thermo Fisher Scientific (4 mentions)
Rpmi 1640, by Nacalai Tesque (3 mentions)
Histopaque 1077, by Merck Group (3 mentions)
Recombinant human gm csf, by R&D Systems (1 mentions)
Antibiotics antimycotic, by Thermo Fisher Scientific (1 mentions)
Neural tissue dissociation kit, by Miltenyi Biotec (1 mentions)
Macs buffer, by Miltenyi Biotec (1 mentions)
Cd11b microbead, by Miltenyi Biotec (1 mentions)
Ls column, by Miltenyi Biotec (1 mentions)
Gm csf, by R&D Systems (1 mentions)
Microglia medium, by ScienCell (1 mentions)
Ifn γ, by R&D Systems (1 mentions)
Interleukin 4 (il 4), by R&D Systems (1 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions)
4 protocols using recombinant human il 34
1
Differentiation of Monocytes into Macrophages and Induced Microglia
2
Isolation of Primary Microglia from Human Tissue
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Human tissue samples were dissociated using a Neural Tissue Dissociation Kit (Miltenyi Biotec, Bergisch-Gladbach, Germany), according to the manufacturer’s instructions (Figure 1
3
In Vitro Differentiation of Human Microglia
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Peripheral blood was collected from patients with MMD and healthy participants into heparin tubes. Peripheral blood mononuclear cells (PBMCs) were separated by density gradient centrifugation in Histopaque-1077 (Sigma Chemical Co., St. Louis, MO, USA); PBMCs were then transferred to RPMI-1640 (Nacalai Tesque, Kyoto, Japan) medium containing 10% heat-inactivated fetal bovine serum. PBMCs were plated in culture chambers at a density of 4 × 105 cells/mL and incubated overnight under standard culture conditions (37 °C, 5% CO2). After overnight incubation, the culture supernatant and non-adherent cells were removed. Adherent cells (monocytes) were cultured in RPMI-1640 Glutamax (Invitrogen) supplemented with 1% antibiotic/antifungal, recombinant human GM-CSF (10 ng/mL; R&D Systems, Minneapolis, USA) and recombinant human IL-34 (100 ng/mL; R&D Systems) for 14 days to generate iMG cells. Then, resting with nothing added, M1 microglia were induced by adding IFNG (100 ng/mL; R&D Systems), whereas M2 were induced with IL-4 (10 ng/mL; R&D Systems), and the two were cultured for another day19 (link),20 (link). Human iMG cells have been validated to be similar to human microglia, using RNA sequencing and cell surface markers19 (link)–24 (link).
4
Differentiation of Monocytes into Induced Microglia
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Peripheral blood was collected using a heparinized tube from healthy volunteers and patients with fibromyalgia. Peripheral blood mononuclear cells (PBMCs) were isolated by Histopaque-1077 (Sigma Chemical Co., St. Louis, MO, USA) density gradient centrifugation. PBMCs were resuspended with RPMI-1640 (Nacalai Tesque, Kyoto, Japan), 10% heat-inactivated fetal bovine serum (FBS; Japan Bio Serum, Hiroshima, Japan), and 1% antibiotic/antimycotic (Invitrogen, Carlsbad, CA, USA). PBMCs were plated onto culture chambers at a density of 4 × 105 cells/ml and cultured overnight in standard culture conditions (37 °C, 5% CO2). After overnight incubation, culture supernatant and non-adherent cells were removed. Adherent cells (monocytes) were cultured with RPMI-1640 Glutamax (Invitrogen) supplemented with 1% antibiotic/antimycotic and recombinant human GM-CSF (10 ng/ml; R&D Systems, Minneapolis, MN, USA) and recombinant human IL-34 (100 ng/ml; R&D Systems) for 14 days to develop the iMG cells14 (link),15 .
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