The supernatants from BMDMs grown in 60 mm culture dishes were collected, and the media were concentrated 50-fold using Amicon ultra-4 centrifugal filter units (Millipore). In some dishes, the cells were treated with Ang II (200 nm; Sigma) for 24 hours or IL4/IL13 (20 ng/mL each, R&D systems) for 48 hours. The concentrated proteins were mixed with equal volume of 2X Laemmli sample buffer (Biorad) containing β-mercaptoethanol and boiled for 8 minutes at 95°C. After centrifugation, 20 μL of the samples were then loaded onto SDS-PAGE (10% wt/vol) and electrophoretically transferred to polyvinylidene fluoride (PVDF) membranes. After blocking, the membranes were probed with antibodies against Tgfβ1 (ab92486;1:2000) and Tgfβ2 (ab36495; 1:2000). Membranes were then incubated with appropriate HRP-conjugated secondary antibodies. Proteins on the membranes were visualized by chemiluminescence detection kit (Super signal west femto maximum sensitivity substrate; Thermo Scientific). The proteins in the media were normalized to total protein contents of the cell extracts.
Il 4 il 13
Manufactured by R&D Systems
Sourced in United States
IL-4 (Interleukin-4) and IL-13 (Interleukin-13) are cytokines that play important roles in immune system regulation. R&D Systems offers these proteins as laboratory research tools for the study of their biological functions.
Automatically generated - may contain errors
Lab products found in correlation
Ang 2, by Merck Group (1 mentions)
Amicon ultra 4 centrifugal filter unit, by Merck Group (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
Epilife, by Thermo Fisher Scientific (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Primary human keratinocyte lines, by Thermo Fisher Scientific (1 mentions)
Human keratinocyte growth supplement s7, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Il 22, by R&D Systems (1 mentions)
Lps ifn γ, by R&D Systems (1 mentions)
Lipopolysaccharides (lps), by R&D Systems (1 mentions)
Lipopolysaccharide lps, by Merck Group (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
Ifn γ, by R&D Systems (1 mentions)
X vivo 15 medium, by Lonza (1 mentions)
4 protocols using il 4 il 13
1
Quantification of TGF-β Proteins
2
Prg4 Deficient BMDM M2a Induction
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The bone marrows of the femurs and tibias of 2-month-old Prg4GT/GT and Prg4+/+ animals were isolated, and BMDMs were generated as described [36 (link), 38 ]. On day 7, M2a BMDMs were induced by IL-4 + IL-13 (20 ng/mL for both cytokines; R&D Systems) for 24h [38 ].
3
Primary Keratinocyte Cytokine Response
4
Stimulating Frozen PBMC Transcriptome
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Frozen human PBMC (ZenBio, Durham, NC, USA) were thawed and resuspended in XVivo 15 medium (Lonza, Verviers, Belgium). 50 µL of 2x10 6 cells/mL (1x10 5 cells/well nal) were treated with 50 µL vehicle control (saline; KD Medical, Columbia, MD, USA) or LMWF5A (Ampio Pharmaceuticals, Englewood, CO, USA) in 96-well plates. Cells were then incubated at 37°C and 5% CO 2 for 1 h.
Immunostimulation was achieved by the addition of 10 µL lipopolysaccharide (LPS; nal concentration of 100 ng/mL; Sigma, St. Louis, MO, USA), 10 µL LPS + IFNγ ( nal concentration of 100 ng/mL LPS and 20 ng/mL IFNγ; R&D Systems, Minneapolis, MN, USA), or 10 µL IL-4 + IL-13 ( nal concentration of 10 ng/mL each; R&D Systems). Cells were further incubated at 37°C and 5% CO 2 for 24 h. Cells were pelleted by centrifugation at 1000 rpm for 10 min. Medium was removed, and cells were processed for RNA isolation using the miRNeasy Mini Kit (Qiagen, Hilden, Germany). Ten technical treatment replicates per plate were combined after Qiazol addition to ensure that a su cient amount of RNA was isolated for downstream analysis. This protocol was repeated three times on different days using a single, representative PBMC donor.
Immunostimulation was achieved by the addition of 10 µL lipopolysaccharide (LPS; nal concentration of 100 ng/mL; Sigma, St. Louis, MO, USA), 10 µL LPS + IFNγ ( nal concentration of 100 ng/mL LPS and 20 ng/mL IFNγ; R&D Systems, Minneapolis, MN, USA), or 10 µL IL-4 + IL-13 ( nal concentration of 10 ng/mL each; R&D Systems). Cells were further incubated at 37°C and 5% CO 2 for 24 h. Cells were pelleted by centrifugation at 1000 rpm for 10 min. Medium was removed, and cells were processed for RNA isolation using the miRNeasy Mini Kit (Qiagen, Hilden, Germany). Ten technical treatment replicates per plate were combined after Qiazol addition to ensure that a su cient amount of RNA was isolated for downstream analysis. This protocol was repeated three times on different days using a single, representative PBMC donor.
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