Concentrated conditioned media of TAMs isolated from axillary tributaries of IBC patients (n = 15) and non-IBC patients (n = 15) were respectively re-diluted with cell culture media specific for each cell line to equal protein content as described before [59 (link)]. SUM149 and MDA-MB-231 cells were seeded at 2.5 × 105 cells/ml in complete culture media of HAM's-F12 containing 5% FBS and DMEM containing 10% FBS in a humidified atmosphere of 5% CO2 at 37 °C, respectively. At 80% confluence, the cells were washed with PBS and seeded in media with 1%FBS and equal protein content of secretion of TAMs isolated from IBC and non-IBC patients. Control cells were seeded in RPMI with 1% FBS and run in parallel at the same conditions for those cells seeded in TAMs-CM. Analogously, the previous steps were performed for incubation in absence or presence of 100 ng/mL rh IL-10 (Immunotools, Friesoythe, Germany) for 24 h or 48 h and SUM149 cells were lysed for qPCR or Western blot, respectively.
Rh il 10
Manufactured by Immunotools
Sourced in Germany
Rh IL-10 is a recombinant human interleukin-10 (IL-10) product. IL-10 is a cytokine that plays a role in the regulation of the immune response.
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Lab products found in correlation
4 protocols using rh il 10
1
Role of TAMs in IBC progression
2
Monocyte and NK Cell Activation for ADCC
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Purified monocyte subsets and NK cells were pre-incubated at 37 °C for 5 hrs with 100 ng/ml LPS (E. coli 0111:B4; Sigma Aldrich), 10 μg/ml R848 (Invivogen), 100 ng/ml rhIL-12 (Immunotools), 100 ng/ml rhIL-15 (R&D Systems), 20 ng/ml rhIFN-γ (Immunotools), 8 μg/ml S100A9 (Origene Technologies) or 100 ng/ml HMGB1 (R&D Systems) before they were used for ADCC assay with BATDA-labeled GM2-coated A549 as target cells at an E:T ratio of 10:1. Freshly isolated CD16− monocytes were cultured in complete IMDM either in the absence or presence of 50 ng/ml rhM-CSF (Immunotools), 50 ng/ml rhTGF-β (R&D Systems) or 50 ng/ml rhIL-10 (Immunotools) at 37 °C for 24 hrs before their ADCC activity was assessed on trastuzumab-coated SKBR3 at a 10:1 E:T ratio.
3
Modulating Cytokine Signaling in ALCL
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ALCL cell lines were obtained from DSMZ, Braunschweig, Germany. For cytokine complementation experiments, recombinant human Interleukin-10 (10 ng/ml, rh IL-10, Immunotools, Friesoythe, Germany) or rhIL-22 (20 ng/ml, Immunotools) were used. For detection of downstream targets, ALCL cells were incubated with TYK2 inhibitors or pan-JAK inhibitors (including 1 µM JAK inhibitor I, Calbiochem, San Diego, CA, USA) for 3 or 6 h and then incubated with IFN-α for 10 min before immunoblot analysis. Description of quantitative RT-PCR, flow cytometry, cytokine arrays and immunohistochemistry, shRNA sources, CRISPR/Cas9 genome editing and murine lymphoma models can be found in Supplementary Methods.
4
A549 cell culture and IL-10 treatment
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A549 cells are human lung ADC alveolar basal epithelial cells, generated in 1972 by Donald Giard, originating primarily from a 58-year-old Caucasian male. In vitro, A549 grow in monolayer and are widely used in research. 3 × 105–106 A549 cells were cultured with 10% FCS containing medium (Gibco F-12 Nut Mix, Thermo Scientific, complemented with 1% penicillin and streptomycin and 1% L-glutamine) or 0.4% FCS (serum starvation) at 37 °C and 5% CO2. To analyse the effect IL-10, A549 cells were cultured for 24–48 h with rhIL-10 (Immunotools, 11340105, Friesoythe, Germany). Cells were collected and used for flow cytometric analysis or RNA extraction.
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