Il 34

Manufactured by BioLegend

Sourced in United States

IL-34 is a recombinant protein that functions as a cytokine. It is involved in the regulation of various cellular processes.

Automatically generated - may contain errors

Lab products found in correlation

Flt3l, by BioLegend (2 mentions) Easysep human monocyte enrichment kit, by STEMCELL (2 mentions) Fetal bovine serum (fbs), by Merck Group (2 mentions) L glutamine, by Fujifilm (1 mentions) Interleukin 7 (il 7), by BioLegend (1 mentions) Il 15, by BioLegend (1 mentions) Interleukin 3 (il 3), by BioLegend (1 mentions) Flag tag (flag), by Thermo Fisher Scientific (1 mentions) Fetal calf serum (fcs), by Merck Group (1 mentions) Interleukin 4 (il 4), by BioLegend (1 mentions) Lipopolysaccharides (lps), by Thermo Fisher Scientific (1 mentions) Dexamethasone (dex), by Merck Group (1 mentions) Ifn γ, by BioLegend (1 mentions) Il 4 il 13, by BioLegend (1 mentions) Ficoll hypaque, by BD (1 mentions) Glutamax, by Thermo Fisher Scientific (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Caco 2 cell, by American Type Culture Collection (1 mentions) Neurobrew, by Miltenyi Biotec (1 mentions) Plx3397, by Selleck Chemicals (1 mentions)

8 protocols using il 34

Monocyte-Derived Macrophage Polarization

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Peripheral blood mononuclear cells (PBMCs) were isolated from buffy-coated blood of healthy blood donors using Ficoll-Hypaque gradient centrifugation (BD Diagnostics, Franklin Lakes, NJ, USA). To isolate monocytes the EasySep Human monocyte enrichment kit without CD16 depletion was used according to manufacturer’s protocols (StemCell Technologies, Vancouver, Canada). PBMCs and monocytes were seeded into 6-well plates and incubated in absence (control) used as controls, or presence of IL-34 or CSF-1 (10 and 50 ng/ml, BioLegend, San Diego, CA, USA) for 1, 6 and 24 h. CSF-1R blocking antibody or IgG1 control (50 ng/ml, R&D Systems, Minneapolis, MN, USA) were added together with IL-34 or CSF-1 and incubated for 6 and 24 h.
Macrophages were generated at day 8 from 5x10⁵ monocytes plated per well in 6 well plates with complete RPMI media supplemented with 50 ng/ml CSF-1 or IL-34 (Biolegend, San Diego, CA, USA). The cells were polarized on day 8 with LPS/IFN-γ (50 ng/ml, BioLegend, San Diego, CA, USA) or IL-4/IL-13 (50 ng/ml, BioLegend, San Diego, CA, USA) into M1 or M2-like monocyte derived macrophages, respectively, for 24 h, and non-polarized monocyte derived macrophages were used as controls.

Zwicker S., Bureik D., Bosma M., Martinez G.L., Almer S, & Boström E.A. (2016). Receptor-Type Protein-Tyrosine Phosphatase ζ and Colony Stimulating Factor-1 Receptor in the Intestine: Cellular Expression and Cytokine- and Chemokine Responses by Interleukin-34 and Colony Stimulating Factor-1. PLoS ONE, 11(11), e0167324.

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Quantifying CSF-1 and IL-34 in Media

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ELISA-based cytokine quantification kits for CSF-1 (Abnova, Taipei City, Taiwan) and IL-34 (BioLegend, CA, USA) secreted in the conditioned media were commercially available.

Pass H.I., Lavilla C., Canino C., Goparaju C., Preiss J., Noreen S., Blandino G, & Cioce M. (2016). Inhibition of the colony-stimulating-factor-1 receptor affects the resistance of lung cancer cells to cisplatin. Oncotarget, 7(35), 56408-56421.

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Culturing mouse bone marrow-derived dendritic cells

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Cell suspensions of mouse bone marrow were prepared by centrifugation of femurs, tibias and humerus with phosphate buffer saline followed by red blood cell lysis and filtering through a sterile 70 μm filter. The cells were suspended in D-MEM with L-Glutamine (Fujifilm Wako Pure Chemical Industries) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, 0.1 mM MEM nonessential amino acids, 55 μM 2-mercaptoethanol and FLT3L 100 ng/mL (#550704; BioLegend) (DC medium). Cells were seeded at 8×10⁶ cells per well in 4 mL of DC medium with ‘IL-34 100 ng/mL (#577608; BioLegend) or nothing’ and ‘niraparib 2.5 μM or dimethyl sulfoxide (DMSO)’ in 6-well plate and cultured at 37°C with 5% CO₂ for 9 days without replating medium.

Nakamura T., Kajihara N., Hama N., Kobayashi T., Otsuka R., Han N., Wada H., Hasegawa Y., Suzuki N, & Seino K.I. (2022). Interleukin-34 cancels anti-tumor immunity by PARP inhibitor. Journal of Gynecologic Oncology, 34(3), e25.

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Stimulation Assays of Immune Cells

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Short-term stimulation assays were performed in 100 µl whole blood with 100 ng/ml CSF1 (Biolegend) and 100 ng/ml LPS Escherichia coli K12 (Invivogen). Longer stimulation assays (48 hours and 7 days) were performed on PBMCs in polypropylene plates in RPMI-1640 media supplemented with 10% FCS (Sigma). The 48 hours stimulation utilised the following immune stimuli: 100 ng/ml CSF1, 100 ng/ml IL-4, 100 ng/ml IFNγ (all Biolegend), 500 nM Dexamethasone (Sigma), 100 ng/ml LPS, 100 ng/ml PAM3CSK, 100 ng/ml FLAG, 100 ng/ml FSL1, and 10⁸ Listeria cells (All Invitrogen). After stimulation, cells were harvested, and stained as above. The 7-day stimulation utilised the following LDCs: CSF1, IL-34, CSF2, CSF3, IL-3, IL-7, IL-15, and FLT3L (All 25 ng/ml, Biolegend). After stimulation, cells were harvested and stained as above.

Combes T.W., Orsenigo F., Stewart A., Mendis A.S., Dunn-Walters D., Gordon S, & Martinez F.O. (2021). CSF1R defines the mononuclear phagocyte system lineage in human blood in health and COVID-19. Immunotherapy Advances, 1(1), ltab003.

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Caco-2 Cell Culture and Stimulation

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Caco-2 cells (American Type Culture Collection, Rockville, MD, USA) were cultured in DMEM supplemented with 10% FBS (Gibco-Brl/Life Technologies, Paisley, UK), 1% NEEA (Gibco-Brl/Life Technologies, Paisley, UK), and 1% Glutamax (Invitrogen, Carlsbad, CA, USA) at 37°C and 5% CO₂. Cells were seeded in 6- or 24-well plates and after attachment for 48 hours the media was changed and the cells were incubated in the absence (controls) or presence of IL-34 and/or CSF-1 (10 ng/ml, BioLegend, San Diego, CA, USA) for 6 hours. Cell lysates from 24-well plate were subjected to RNA-isolation and cell lysates and supernatants from 6 well plates were subjected to protein analysis.

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Microglial Activation and Signaling Pathways

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Cells were cultured in Dulbecco’s modified Eagles medium (DMEM, Sigma) conditioned with Fetal bovine serum 10% (vol/vol) (FBS, Sigma) and Penicillin/Streptomycin 1% (vol/vol) (Sigma) and M-CSF 20 ng/ml (R&D) or IL-34 (Biolegend). CSF1R was inhibited by PLX3397 10 ng/ml (Selleckchem) and ERK1/2 were inhibited using SCH772984 (Selleckchem) 3 ng/ml. For in vitro assays and transcriptome analysis microglial cells were sorted from single-cell suspensions by Flow cytometry employing the protocol described above or by magnetic beads. By Flow cytometry, cells from mouse CNS were sorted using a BD Influx cell sorter. Microglia were sorted as live CD11b⁺ CD45^Intermediate Ly6G⁻ with enhanced yellow fluorescent protein (eYFP⁺) (Fig. S1) followed by P-ERK 1/2 and P-Akt gating (Fig. 3). For bulk microglia sorting in naïve mouse CNS, we used CD11b magnetic beads and column magnetic cell sorter (MACS, Miltenyi Biotec). Neuronal cells for co-cultures were established from cells isolated P5 mouse CNS and cultured with Neurobrew (Miltenyi biotec) for 14 days before microglia were added.

Berglund R., Cheng Y., Piket E., Adzemovic M.Z., Zeitelhofer M., Olsson T., Guerreiro-Cacais A.O, & Jagodic M. (2024). The aging mouse CNS is protected by an autophagy-dependent microglia population promoted by IL-34. Nature Communications, 15, 383.

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Quantifying Cytokine Levels in CSF-1 and IL-34

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Cytokine quantification levels of CSF-1 (Abnova, Taipei City, Taiwan) and IL-34 (BioLegend) secreted in the conditioned media (72 h) were quantitated by ELISA.

Cioce M., Canino C., Goparaju C., Yang H., Carbone M, & Pass H.I. (2014). Autocrine CSF-1R signaling drives mesothelioma chemoresistance via AKT activation. Cell Death & Disease, 5(4), e1167-.

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Monocyte-Derived Macrophage Generation

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PBMCs (peripheral blood mononuclear cells) were isolated from buffy-coated blood using Ficoll-Hypaque gradient centrifugation (BD Diagnostics), followed by monocyte isolation using the EasySep Human monocyte enrichment kit without CD16 depletion (StemCell Technologies), according to the manufacturers’ protocols. Then, 5×10⁵ monocytes/well were plated in six-well plates with complete RPMI 1640 medium supplemented with 50 ng/ml CSF-1 or IL-34 (BioLegend) for 8 days to generate macrophages.

Zwicker S., Martinez G.L., Bosma M., Gerling M., Clark R., Majster M., Söderman J., Almer S, & Boström E.A. (2015). Interleukin 34: a new modulator of human and experimental inflammatory bowel disease. Clinical Science (London, England : 1979), 129(Pt 3), 281-290.

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