5 × 105/ml FLS (n = 5) in 10 cm dish were incubated with FBS-free DMEM for 24 hrs, then stimulated with or without IL-34 (50 ng/ml, R&D Systems, USA) for another 24 h, which were prepared for the detection of IL-6 mRNA expression. FLS (1 × 105/ml) were starved in 6-well plate for 24 h, then stimulated with or without IL-34 (50 ng/ml) for 12, 24, 48, and 72 h (n = 6) or pretreated with anti-CSF-1R antibody (25 ng/ml, R&D Systems, USA) for 30 min (n = 8). The supernatants of cell culture were collected to measure the levels of IL-6.
Il 34
Manufactured by R&D Systems
Sourced in United States
IL-34 is a recombinant human protein that functions as a cytokine. It is a member of the interleukin family of proteins.
Automatically generated - may contain errors
Lab products found in correlation
Gm csf, by R&D Systems (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Molt 4, by RIKEN Cell Bank (1 mentions)
Sb203580, by Merck Group (1 mentions)
Anti β actin antibody, by Merck Group (1 mentions)
Recombinant ptx3, by R&D Systems (1 mentions)
Il lβ, by R&D Systems (1 mentions)
Pd98059, by Merck Group (1 mentions)
Il 17, by R&D Systems (1 mentions)
Du145, by American Type Culture Collection (1 mentions)
Il 23, by R&D Systems (1 mentions)
Anti m csf, by R&D Systems (1 mentions)
Anti gm csf, by R&D Systems (1 mentions)
Wallac 1420 victor2, by PerkinElmer (1 mentions)
M csf, by R&D Systems (1 mentions)
Cell proliferation reagent wst 1, by Roche (1 mentions)
Protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
8 protocols using il 34
1
IL-34 Regulation of IL-6 in FLS
2
Investigating Macrophage TNF-α Production
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Bone marrow derived macrophages (BMMΦs) were isolated from 6- to 8-week-old C57BL/6 mice as previously described [1 (link)] and cultured with 10 ng/mL GM-CSF (R&D) for 7 days to drive M1 macrophage maturation. The mouse macrophage cell line, RAW264.7, was also treated with GM-CSF (10 ng/mL) for 2 days to induce M1-like macrophages. All cells were maintained at 37°C in a 5% (v/v) CO2 enriched atmosphere in RPMI 1640 medium, supplemented with 10% (v/v) fetal bovine serum (FBS) containing penicillin and streptomycin.
To investigate TNFα production by mouse macrophages, mature M1 BMMΦs or RAW264.7 cells were first seeded at levels of 2 × 105 cells in the wells of a 24-well tissue culture plate. To these cells, IL-34 (R&D Systems) was added at 0, 10, or 100 ng/mL, and cells were then incubated with the added presence of 10 ng/mL GM-CSF for 5 (mature M1 BMMΦs) or 2 (RAW264.7 cells) days. After incubation, 1 × 105 HKC, which had been prepared as previously described [1 (link), 13 (link)], was added. Wells devoid of HKC served as negative controls. Cell culture supernatants were collected 24 h after stimulation and TNFα was detected using an enzyme-linked immunosorbent assay (ELISA).
To investigate TNFα production by mouse macrophages, mature M1 BMMΦs or RAW264.7 cells were first seeded at levels of 2 × 105 cells in the wells of a 24-well tissue culture plate. To these cells, IL-34 (R&D Systems) was added at 0, 10, or 100 ng/mL, and cells were then incubated with the added presence of 10 ng/mL GM-CSF for 5 (mature M1 BMMΦs) or 2 (RAW264.7 cells) days. After incubation, 1 × 105 HKC, which had been prepared as previously described [1 (link), 13 (link)], was added. Wells devoid of HKC served as negative controls. Cell culture supernatants were collected 24 h after stimulation and TNFα was detected using an enzyme-linked immunosorbent assay (ELISA).
3
Establishing Human T-cell Leukemia Cell Lines
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The human ATLL cell lines, ATL-T and ED, were previously established by Taylor and
Matsuoka.22 (link) The T-cell leukemia cell
line MOLT-4 was purchased from the RIKEN Cell Bank (WAKO, Japan). Control TF-1 cells
(TF-1CT) and TF-1 cells expressing M-CSFR
(TF-1MCSFR) were previously established by Suzu.23 (link) Cells were maintained in RPMI1640
supplemented with 10% fetal bovine serum. Mycoplasma testing was performed using a
polymerase chain reaction (PCR) detection kit (Takara Bio Inc., Otsu, Japan). Conditioned
medium (CM) from cell lines was obtained as described previously.24 (link) Recombinant M-CSF was a gift from Morinaga Milk Industry
(Kanagawa, Japan). IL-34 was purchased from R&D Systems (Minneapolis, MN, USA).
PLX3397 (pexidartinib) was obtained from APExBio (Houston, TX, USA). Cell block samples of
cell lines were prepared as described previously.25 (link)
Matsuoka.22 (link) The T-cell leukemia cell
line MOLT-4 was purchased from the RIKEN Cell Bank (WAKO, Japan). Control TF-1 cells
(TF-1CT) and TF-1 cells expressing M-CSFR
(TF-1MCSFR) were previously established by Suzu.23 (link) Cells were maintained in RPMI1640
supplemented with 10% fetal bovine serum. Mycoplasma testing was performed using a
polymerase chain reaction (PCR) detection kit (Takara Bio Inc., Otsu, Japan). Conditioned
medium (CM) from cell lines was obtained as described previously.24 (link) Recombinant M-CSF was a gift from Morinaga Milk Industry
(Kanagawa, Japan). IL-34 was purchased from R&D Systems (Minneapolis, MN, USA).
PLX3397 (pexidartinib) was obtained from APExBio (Houston, TX, USA). Cell block samples of
cell lines were prepared as described previously.25 (link)
4
Breast and Prostate Cancer Cell Lines
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MCF-7 and MDA-MB-231 human breast cancer cell lines and DU-145 and PC-3 human prostate cancer lines were from American Type Culture Collection (Manassas, VA, USA) and were maintained according to distributors ' instructions. All cells were grown in a 5% CO2 atmosphere at 37°C. Pyrrolidine dithiocarbamate (PDTC), SP600125, SB203580, PD98059, and anti-β-actin antibody were purchased from Sigma (St. Louis, MO, USA). Recombinant PTX3, IL-lβ, IL-17, IL-23, IL-34 and TNFα proteins and rabbit anti-human PTX3 antibody for ELISA were from R&D Systems (Minneapolis, MN, USA).
5
Culturing Immunopanned Microglia with IL-34
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For the in vitro experiments, immunopanning-isolated microglia were seeded in 96-well plates at 5x104 cells/well, and cultured (at 37°C, 5% CO2) for 7 days in DMEM/F12 supplemented with 10% HI-FCS, 100 units(μg)/mL penicillin/streptomycin and 1x MEM-NEAAs in addition to 40 ng/mL IL-34 (R&D SYSTEMS, #5265-IL-010). No media changes were performed for the duration of the experiments. The culture was supplemented with 40 ng/mL IL-34 every second day (days 2, 4 and 6).
6
Monocyte Viability Assay with Cytokines
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After thawing, CD14+ monocytes were counted and resuspended in αMEM medium supplemented with 20% FBS. In a 96-well plate, we added 50 μL of cells in each well (45,000 cells/well) and then 50 μL of M-CSF, IL-34, and GM-CSF (all from R&D systems) diluted in 2% RPMI to obtain a 25, 50, and 10 ng/mL final concentration, respectively, or 50 μL of RA SF conditioned media. Medium (50 μL; 2% FBS) without any cytokine was used as control condition (CT). After 3 days, cell viability was assessed by WST-1 cell proliferation Reagent (Roche, France) by adding 10 μL per well. The formazan dye obtained from WST-1 cleavage by mitochondrial dehydrogenase was read by a microplate reader Wallac 1420 Victor 2 (Perkin Elmer, USA) between 420 and 480 nm after incubation for 4 hours at 37°C. Antibodies used to identify cytokines implicated in monocyte viability were as follows: anti M-CSF (R&D Systems), anti-IL-34 (Diaclone, INSERM UMR957), and anti-GM-CSF (R&D Systems) at 2, 10, and 5 μg/mL, respectively. We have previously tested different antibody concentrations to determine the most appropriate concentrations.
7
N13 Murine Microglial Cell Stimulation
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The N13 murine microglia cell line (21 (link)) was cultured in Dulbecco's modified Eagle's medium (DMEM, Thermo Fisher Scientific), supplemented with 10% fetal bovine serum and 50 U/mL penicillin/ streptomycin (Thermo Fisher Scientific). Cells were maintained in T75 flasks at 37°C in a 5% CO2 humidified atmosphere. Cells were plated at a density of 2 × 105 cells/cm2 in 6-well-plates and cultured overnight to allow adherence. Cells were plated at a density of 2 × 105 cells/cm2 in 6-well-plates and cultured overnight to allow adherence. Cells were kept in serum-free medium for 4 h prior to stimulation and then incubated for the indicated time points (5 or 10 min) with recombinant CSF-1 (50 or 100 ng/mL), IL-34 (50 or 100 ng/mL) (R&D Systems) or LPS (1 μg/mL) as a negative control for CSF1R pathway activation (22 (link), 23 (link)), after which cells were immediately lysed in RIPA buffer (Thermo Fisher Scientific), supplemented with protease and phosphatase inhibitor cocktails (Roche, Thermo Fisher Scientific). Protein lysates were concentrated using Microcon-10kDa Centrifugal Filter Units (Merck Millipore), according to manufacturer's instructions and protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Protein lysates were subjected to SDS-PAGE and Western blot.
8
Cytokine and Receptor Modulation
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Recombinant mouse IL-1β, TNF-α, and IL-34 were purchased from R&D Systems (Minneapolis, MN, USA). The c-fms/CSF1R tyrosine kinase inhibitor GW2580 was used as a blocker of CSF1R signaling (Millipore, Bedford, MA, USA). Dylight 594–labeled tomato lectin was used as a capillary endothelial cell marker (Vector Laboratories, Burlingame, CA, USA).
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