To obtain bone marrow-derived dendritic cells (BMDCs), seven-to-eight-week-old BALB/c mice were anaesthetised with an intraperitoneal injection of ketamine (80 mg/kg body weight) and xylazine (10 mg/kg body weight) and were euthanised by cervical dislocation (n = 5 mice). Bone marrow was removed from femur and tibia after mouse death by flushing into complete RPMI (Life Technologies), as previously described.17 (link) Eluted cells were cultured at 37 °C in a controlled humidified atmosphere with 5% CO2 for 5 days in complete RPMI and 20 ng/mL IL-4 and GM-CSF (R&D Systems, Minneapolis, MN), until approximately 50% of cells were CD11c+.
Gm csf and il 4
Manufactured by R&D Systems
Sourced in United States
GM-CSF and IL-4 are recombinant human cytokines produced in E. coli. GM-CSF stimulates the production and function of granulocytes and macrophages, while IL-4 induces the differentiation of naive helper T cells into Th2 cells.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Complete rpmi, by Thermo Fisher Scientific (1 mentions)
Polyethylenimine (pei), by Polysciences (1 mentions)
Dynabeads cd4 positive isolation kit, by Thermo Fisher Scientific (1 mentions)
Protein a sepharose column, by Abcam (1 mentions)
Monocyte purification kit, by Miltenyi Biotec (1 mentions)
Hrp conjugated goat anti human igg h l, by Jackson ImmunoResearch (1 mentions)
Human pbmcs, by AllCells (1 mentions)
Anti human cd28 clone cd28.2, by BioLegend (1 mentions)
Matrigel, by BD (1 mentions)
Ficoll gradient, by GE Healthcare (1 mentions)
Sypro orange, by Merck Group (1 mentions)
Freestyle 293 medium, by Thermo Fisher Scientific (1 mentions)
Cytotoxicity detection kit ldh, by Roche (1 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (1 mentions)
Tmb substrate, by Thermo Fisher Scientific (1 mentions)
Ficoll paque, by GE Healthcare (1 mentions)
Cd14 microbead, by Miltenyi Biotec (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Ficoll hypaque gradient, by GE Healthcare (1 mentions)
11 protocols using gm csf and il 4
1
Isolation of Murine Bone Marrow-Derived Dendritic Cells
2
Comprehensive Cell Line Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NCI-N87, HCC1954, and SKBR3 cells were obtained from the Shanghai Cell Line Bank. RPMI1640 medium (61870036), Freestyle 293 medium, Dynabeads CD4 Positive Isolation Kit (11331D), TMB substrate, and fetal calf serum (FCS) were obtained from Thermo Fisher Scientific. PE-labeled goat anti-human IgG (409304), streptavidin-PE (405203), anti-human-CD3 (clone HIT3a), and anti-human-CD28 (clone CD28.2) were obtained from Biolegend. Trastuzumab and humanized anti-PD-L1 scFV were prepared in house. Cell Counting Kit-8 (CCK8) was obtained from Dojindo Laboratories. Human antibody germline sequence and primers were synthesized by Sangon Biotech. IL-4 and GM-CSF were obtained from R&D Systems, and all other recombinant proteins were products of Sino Biological. SYPRO Orange was obtained from Sigma-Aldrich. Human IFN-γ ELISA Kit (EHC102g) was obtained from Neobioscience. Cytotoxicity detection kit (LDH) was from Roche Life Science. Matrigel was obtained from BD Biosciences. Polyethylenimine (PEI) was obtained from Polysciences. Protein A probes were obtained from Pall Corporation. Protein A-Sepharose Column was obtained from BioVision. HRP-conjugated goat anti-human IgG (H + L) was obtained from Jackson ImmunoResearch. Monocyte purification kit was obtained from Miltenyi Biotec. Ficoll gradient was obtained from GE healthcare. Human PBMCs were obtained from Allcells.
3
Bone Marrow Dendritic Cell Culture
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Freshly isolated bone marrow cells from femur and tibia of B6 or immunized mice were cultured in RPMIs medium supplemented with 100 μg/mL penicillin and streptomycin mixture, 2 mM l -glutamine, 50 μM β-mercaptoethanol, 10% heat-inactivated filtered fetal calf serum, and 10 ng/ml of recombinant murine GM-CSF and IL-4 (10 ng/ml) (R&D Systems) for 5 days at 37 °C in a 5% CO2 incubator, as described previously (Inaba et al., 1992 (link)).
4
Generating Bone Marrow Dendritic Cells for T Cell Activation
5
Bone Marrow Dendritic Cell Differentiation and Functional Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bone marrow dendritic cells were generated by incubating bone marrow cells for 5 days in the presence of 10 ng/ml of recombinant murine GM-CSF and IL-4 (R&D Systems), as described previously (35 (link)). Cytokine (IL-1β, IL-6, L-12 and IL-23) levels in the culture medium were measured by ELISA.
To determine the antigen-presenting function, BMDCs were incubated in a 24-well plate with responder T cells isolated from immunized B6 mice under Th1- or Th17-polarizing conditions. Forty-eight hours after stimulation, IFN-γ and IL-17 in the culture medium were measured by ELISA. The percentage of IFN-γ+ and IL-17+ T cells among the responder T cells was determined by intracellular staining after 5 days of culture as described above.
To determine the antigen-presenting function, BMDCs were incubated in a 24-well plate with responder T cells isolated from immunized B6 mice under Th1- or Th17-polarizing conditions. Forty-eight hours after stimulation, IFN-γ and IL-17 in the culture medium were measured by ELISA. The percentage of IFN-γ+ and IL-17+ T cells among the responder T cells was determined by intracellular staining after 5 days of culture as described above.
6
Bone Marrow Dendritic Cell Generation and Cytokine Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bone marrow dendritic cells (BMDCs) were generated by incubation of bone marrow cells for 5 days in the presence of 10 ng/ml of recombinant murine GM-CSF and IL-4 (R&D Systems), as described previously [42 (link)]. In this study, the BMDCs were generated and cultured with or without oxATP (80 μM). Cytokine (IL-1, IL-6, L-12 and IL-23) levels in the culture medium were measured by ELISA. To determine antigen-presenting function, BMDCs were incubated in a 24-well plate with responder T cells isolated from immunized B6 mice under Th1- or Th17-polarizing conditions. Forty-eight hours after stimulation, IFN-γ and IL-17 in the culture medium were measured by ELISA. The percentage of IFN-γ+ and IL-17+ T cells among the responder T cells was determined by intracellular staining after 5 days of culture as described above.
7
Generation and Characterization of BMDCs
8
Generating Immature Dendritic Cells from Monocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Peripheral blood mononuclear cells (PBMCs) were isolated using the Ficoll-Paque method (GE Healthcare Life Sciences, Piscataway, NJ) from buffy coats. CD14+ monocytes were isolated from PBMCs using MidiMACS Technology with CD14 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Next, CD14+ monocytes were cultured at 1 × 106 cells/ml in Roswell Park Memorial Institute (RPMI)-1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco) in the presence of GM-CSF and IL-4 (50 ng/ml and 35 ng/ml; R&D Systems, Minneapolis, MN, USA) at 37°C and 5% CO2 for 7 days. On day 3, half of the medium was removed from culture and replenished with the same volume of fresh medium containing twofold concentrations of GM-CSF and IL-4. On day 5, the same step was repeated. On day 7, the imDCs were ready for experimental use.
9
Generation and Characterization of BMDCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BMDCs were generated by incubation of bone marrow cells for 5 days in the presence of 10 ng/mL of recombinant murine GM-CSF and IL-4 (R&D Systems), as described previously (Inaba et al., 1992 (link)). Cytokine (IL-1, IL-6, l -12 and IL-23) levels in the culture medium were measured by ELISA. To determine antigen-presenting function, BMDCs were incubated in a 24-well plate with responder T cells isolated from immunized B6 mice under Th1- or Th17-polarizing conditions. Forty-eight hours after stimulation, IFN-γ and IL-17 in the culture medium were measured by ELISA. The percentage of IFN-γ+ and IL-17+ T cells among the responder T cells was determined by intracellular staining after 5 days of culture as described above.
10
Generation of Tumor-Associated Dendritic Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Peripheral blood mononuclear cells (PBMCs) of healthy donors were isolated using Ficoll-Hypaque gradient (GE Healthcare Bio-Sciences, Little Chalfont, UK), and CD14+ monoclonal antibody-conjugated magnetic beads (MACS MicroBeads; Miltenyi Biotec Ltd, Bergisch Gladbach, Germany) were used for CD14+ monocyte isolation from PBMCs, following the manufacturer’s instructions. CD14+ monocytes were cultured in RPMI-1640 medium containing 50% L-15 (for control) or SW620-CM (for TADCs), in the presence of 10 ng/mL GM-CSF and IL-4 (R&D Systems, Minneapolis, MN, USA) for five days to generate monocyte-derived DCs or TADCs. The study protocol was approved by the Institutional Review Board (IRB) of Kaohsiung Medical University Hospital (Kaohsiung, Taiwan), and all participants provided written informed consent in accordance with the Declaration of Helsinki before entering the study.
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!