All ELISAs with the exception of the hIL-18 and mIL-18 ELISA (MBL International antibodies D044-3, D045-6 and Thermofisher #BMS267-2, respectively) were from R&D Systems (#DY210, DY201, DY401, DY400, DY200). Quantification of cell death was performed by analysis of LDH release in the cell supernatant. Cell death was also quantified by monitoring propidium iodide (PI) incorporation. Briefly, 3 to 5 × 104 U937 cells, hMDMs or BMDMs were seeded in 0.3 cm2 wells of a black 96-flat-bottom-well plate. One hour after infection, PI was added at a final concentration of 5 μg.mL−1. Fluorescence was measured over a 24 h period on a microplate fluorimeter (Tecan).
D044 3
Manufactured by R&D Systems
Sourced in United States
The D044-3 is a lab equipment product from R&D Systems. It is a tool used for scientific research and analysis, but a detailed description of its core function cannot be provided while maintaining an unbiased and factual approach. Additional information is not available.
Automatically generated - may contain errors
Lab products found in correlation
D045 6, by R&D Systems (2 mentions)
Dy210, by R&D Systems (1 mentions)
Microplate fluorimeter, by Tecan (1 mentions)
Af 210 na, by R&D Systems (1 mentions)
Af717, by R&D Systems (1 mentions)
Af 200 na, by R&D Systems (1 mentions)
Ab 108 c, by R&D Systems (1 mentions)
Mab002, by R&D Systems (1 mentions)
Af840, by R&D Systems (1 mentions)
Mab840, by R&D Systems (1 mentions)
3 protocols using d044 3
1
Quantification of Cell Death in Immune Cells
2
Cytokine Neutralization and Antimicrobial Peptide Assay
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The cells were seeded in exactly the same way as described above for coculture experiments. After overnight attachment of the cells, neutralizing antibodies or isotype controls (100 ng/ml) were added to the cultures as recommended by the supplier. Neutralizing antibodies against IL-1α (R&D: AF-200-NA), TNF-α (R&D: AF-210-NA), CCL27 (R&D: AF-376), and CCL28 (R&D: AF-717) all had goat IgG (R&D: AB-108-C) as an isotype control. For IL-18 (R&D: D044-3), a mouse IgG1 (R&D: MAB002) was used as an isotype control. After 30 min, the culture medium was further supplemented with Hst1 (4 and 50 μM), LL-37 (2 and 4 μM), or vehicle (H2O). 24 h after exposure, the supernatant was collected and stored at −20°C until further analysis by ELISA and cell viability was determined using the MTT assay (as described above).
3
Cytokine and Protease Profiling in Cell Culture
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Prolactin, IL1 family members and vascular endothelial cell growth factor (VEGF), metallopeptidase inhibitor 3 (TIMP-3), matrix metallopeptidase 9 (MMP-9), monocyte chemoattractant protein-1 (MCP1) and macrophage migration inhibitory factor (MIF) were measured in culture supernatants, total cell protein extracts, or both. All enzymelinked immunosorbent assay (ELISA) experiments included a standard curve. DuoSet kits (R&D Systems, Minneapolis, MN, USA) were used to assess prolactin, IL33 and its receptor (ST2). IL18 and IL18R1 ELISAs were made using mouse monoclonal anti-human IL18 and rat monoclonal biotin-labelled antihuman IL18 antibodies (D044-3 R&D Systems and D045-6 R&D Systems, Minneapolis, MN, USA, respectively) as well as mouse monoclonal anti-human IL18R1 and goat polyclonal antihuman IL18R1 antibodies (MAB840 R&D Systems and AF-840 R&D Systems, Minneapolis, MN, USA, respectively). IL1 receptor and antagonist, RANTES, VEGF, TIMP-3, MMP-9, MCP1 and MIF ELISAs were carried out as described previously (Bourdiec et al., 2013a (Bourdiec et al., , 2013b;; Veillat et al., 2010 Veillat et al., , 2012)) .
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