BMDMs were seeded in 12-well plates at a density of 5 × 105 cells per well. The cells were polarized into M1 with 50 ng/ml LPS (Sigma-Aldrich, St. Louise, Missouri, USA) + 100 ng/ml IFNγ (Bio-Techne, Minneapolis, Minnesota, USA) or M2 with 20 ng/ml IL-4. (Bio-Techne, Minneapolis, Minnesota, USA) The supernatants were collected 24 h after cytokine stimulation and nitric oxide (NO) was measured using the Griess Reagent System (Promega, Madison, Wisconsin, USA), according to manufacturer’s instructions. IL-1β in BMDM supernatants was measured using the mouse IL-1 beta/IL-1F2 DuoSet ELISA kit (DY401, Bio-Techne, Minneapolis, Minnesota, USA) according to manufacturer’s instructions. Cells were collected for flow cytometry, protein or RNA extraction.
Interleukin 4 (il 4)
Manufactured by Bio-Techne
Sourced in United States, Germany
IL-4 is a recombinant human cytokine produced in E. coli. It is a regulatory T cell-derived cytokine that plays a key role in the differentiation of naive T cells into Th2 effector cells.
Automatically generated - may contain errors
Lab products found in correlation
Lipopolysaccharides (lps), by Merck Group (4 mentions)
M csf, by Bio-Techne (2 mentions)
Gm csf, by Bio-Techne (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Griess reagent system, by Promega (1 mentions)
6 well plate, by Thermo Fisher Scientific (1 mentions)
Human il 6, by Thermo Fisher Scientific (1 mentions)
Rbc lysis buffer, by Thermo Fisher Scientific (1 mentions)
M csf, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Ab serum, by Valley Biomedical (1 mentions)
Classical monocyte isolation kit, by Miltenyi Biotec (1 mentions)
Hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
Anti gapdh, by Merck Group (1 mentions)
Pan t cell isolation kit, by Miltenyi Biotec (1 mentions)
Heparin sulphate, by Merck Group (1 mentions)
Annexin 5 pi, by BD (1 mentions)
Aim 5 media, by Thermo Fisher Scientific (1 mentions)
6 protocols using interleukin 4 (il 4)
1
Macrophage Polarization and Cytokine Assays
2
Microglia Isolation and Polarization Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Microglia were cultured from 4 to 6 week old Socs3fl/fl and DKO mice using a previously described protocol [43 (link)]. Briefly, smashed brain tissue suspensions were filtered through a 70 μm cell strainer (BD Falcon, BD Biosciences). Then the cell suspensions were centrifuged at 600 g for 8 min. The cell pellet was re-suspended in media containing 10% fetal calf serum (FCS), 20 ng/ml M-CSF (Bio-techne, Minneapolis, Minnesota, US), 1% penicillin/streptomycin, 1 mM glutamine in DMEM/F12 (all from Thermo Fisher Scientific, Waltham, MA, USA), and seeded in a 6-well plate (Thermo Fisher Scientific). Floating cells were removed 3 days later and media were changed every 3 days until cells reached 90% confluence. The phenotype of the cells was confirmed by their CD11b and IBA-1 expression (> 90%). The microglia were then treated with 1) M1, or pro-inflammatory stimuli by adding 100 ng/ml IFN-γ (Bio-Techne) and 50 ng/ml LPS (Sigma-Aldrich) or 2) M2, or anti-inflammatory stimuli by adding 20 ng/ml IL-4 (Bio-Techne) overnight. The cells were collected for real-time RT-PCR for cytokine/chemokine gene expression and the supernatants were collected for Luminex assay.
3
Murine and Human Macrophage Differentiation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Murine bone marrow (BM) was flushed from the femur and tibia using a syringe and needle, and RBCs were lysed using RBC lysis buffer (eBioscience). Complete murine BM, or splenic murine Ly6C+ cells or PBMC-derived human CD14+ MACS-sorted monocytes were plated in RPMI 10% FCS, 1× penicillin/streptomycin (Sigma-Aldrich), 50 ng/ml recombinant murine (Bio-techne) or human (Peprotech) M-CSF at 1 × 106 cells/well on 6-well plates for subsequent mRNA and protein analyses. Where viable macrophages were required for ongoing experiments, cells were plated at 5.5 × 106 cells on 6 cm non-tissue culture-treated plates. Additional murine cytokines, IL-4 (Bio-techne) and IFN-γ (Bio-techne), human IL-6 (Peprotech), or LPS (Sigma-Aldrich) were added where indicated in the figure legends at 50 ng/ml unless stated otherwise. After 72 h in culture, macrophage purity was assessed by flow cytometry. Macrophages differentiated in the presence of M-CSF only are referred to as M0 cells, and macrophages differentiated in the presence of M-CSF and IL-6 as M(IL-6) cells.
4
KRAS Sequencing and Monocyte Differentiation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lines and normal cells were purchased from the suppliers listed in supplementary table S6 and cultured in media recommended by the supplier. KRAS (exon 2 region) was sequenced from PCR products amplified from gDNA isolated using QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany) at Eurofins Genomics (Ebersberg, Germany).
PBMCs were either purchased cryo-frozen from commercial suppliers (Cellular Technology Limited (CTL), OH & Tissue Solutions, Glasgow, UK) or prepared from healthy donor leukopaks (ALLCELLS, CA) by Ficoll density gradient centrifugation. CD14 + monocyte cells isolated by negative selection from PBMCs using Classical Monocyte Isolation Kit (Miltenyi, North Rhine-Westphalia, Germany) were differentiated for 4–6 days in Gibco™ AIM V Medium (Thermo Fisher Scientific, MA) with Penicillin-Streptomycin (Gibco™), 5% AB serum (Valley Biomedical, VA), 1000 U/ml GM-CSF and 500 U/ml IL-4 (Biotechne, MN) to generate iDC. Where relevant, T cells were isolated from PBMC by immunopurification using a Pan T Cell Isolation Kit (Miltenyi) according to the manufacturer’s instructions. Rabbit anti-RAS, –RASG12D and HRP-conjugated secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA). Anti-GAPDH was purchased from Merck Millipore (MA). Purified synthetic peptides were purchased from Peptide Protein Research Ltd, UK
PBMCs were either purchased cryo-frozen from commercial suppliers (Cellular Technology Limited (CTL), OH & Tissue Solutions, Glasgow, UK) or prepared from healthy donor leukopaks (ALLCELLS, CA) by Ficoll density gradient centrifugation. CD14 + monocyte cells isolated by negative selection from PBMCs using Classical Monocyte Isolation Kit (Miltenyi, North Rhine-Westphalia, Germany) were differentiated for 4–6 days in Gibco™ AIM V Medium (Thermo Fisher Scientific, MA) with Penicillin-Streptomycin (Gibco™), 5% AB serum (Valley Biomedical, VA), 1000 U/ml GM-CSF and 500 U/ml IL-4 (Biotechne, MN) to generate iDC. Where relevant, T cells were isolated from PBMC by immunopurification using a Pan T Cell Isolation Kit (Miltenyi) according to the manufacturer’s instructions. Rabbit anti-RAS, –RASG12D and HRP-conjugated secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA). Anti-GAPDH was purchased from Merck Millipore (MA). Purified synthetic peptides were purchased from Peptide Protein Research Ltd, UK
5
Monocyte-derived Dendritic Cell Culture
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Blood packs were acquired from the National Health Service Blood and Transplant Service (NHSBTS) an HTA licenced provider. Monocytes were isolated using CD14 microbeads (Miltenyi Biotec) and the MACS system (Mitenyi Biotec). After treatment with 1500U/ml IL4 (Bio-Techne) and 400U/ml GMCSF (Bio-Techne), cells were cultured in AIM-V media (GIBCO) for 6 days. Cells were confirmed to be immature DCs using flow cytometry as described27 (link). Extract was diluted in PBS to reach stock concentration for use on cells. Heparin sulphate (Sigma) was dissolved in PBS before use. Cell viability was assayed using Annexin-V/PI (BD biosciences) staining by flow cytometry.
6
Microglia Isolation and Polarization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Microglia were cultured from 4-6 week old Socs3 / and DKO mice using a previously described protocol (44) . Brie y, smashed brain tissue suspensions were ltered through a 70 μm cell strainer (BD Falcon, BD Biosciences). Then the cell suspensions were centrifuged at 600g for 8 min. The cell pellet was re-suspended in media containing 10% fetal calf serum (FCS), 20ng/ml M-CSF (Bio-techne, Minneapolis, Minnesota,US), 1% penicillin/streptomycin, 1 mM glutamine in DMEM/F12 (all from Thermo Fisher Scienti c, Waltham, MA, USA), and seeded in a 6-well plate (Thermo Fisher Scienti c). Floating cells were removed 3 days later and media were changed every 3 days until cells reached 90% con uence. The phenotype of the cells was con rmed by their CD11b and IBA-1 expression (>90%).The microglia were then treated with 1) M1, or pro-in ammatory stimuli by adding 100ng/ml IFN-γ (Bio-Techne)and 50ng/ml LPS (Sigma-Aldrich)or 2) M2, or anti-in ammatory stimuli by adding 20ng/ml IL-4 (Bio-Techne) overnight. The cells were collected for real-time RT-PCR for cytokine/chemokine gene expression and the supernatants were collected for Luminex assay.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!