Bone marrow-derived dendritic cells (BMDCs) were generated according to a modified version of a previously described method(Helft et al., 2015 (link); Lutz et al., 1999 (link)). Briefly, bone marrow cells isolated from the femurs and tibia of 7–14-week-old WT mice were filtered through a 70-μm nylon strainer, red blood cells lysed by ACK lysis buffer (Sigma Aldrich), and cultured in BMDC medium (RPMI-1640 containing 10% FBS, 100 U/mL PS, 2 mM l -glutamine (Gibco), 50 μM 2-mercaptoethanol (Sigma Aldrich), 20 ng/mL GM-CSF (PeproTech), and 10 ng/mL IL-4 (PeproTech)). The BMDC medium was replaced on days 3 and 6. On day 8, non-adherent cells were harvested and BMDC purity was assessed by flow cytometry to ensure staining for markers CD11c, MHC II, CD11b, and CD86. For DC vaccination, non-adherent cells were pulsed for 3 h at 37°C with 10 μM of the human gp10025–33 (hgp100) peptide (GenScript) in Opti-MEM media (Gibco) and washed three times with PBS before their use. For induction of vitiligo in mice, 1.0×106 CD11c+MHC II+ hgp100 pulsed BMDCs were co-injected intravenously into WT, IFNaR-deficient or IFNGR-deficient hosts.
Human gp10025 33 hgp100 peptide
Manufactured by GenScript
Human gp10025–33 (hgp100) peptide is a synthetic peptide corresponding to a specific amino acid sequence within the human glycoprotein 100 (gp100) protein. It is commonly used in research applications, particularly in the study of immune system responses and related phenomena.
Automatically generated - may contain errors
Lab products found in correlation
Interleukin 4 (il 4), by Thermo Fisher Scientific (2 mentions)
Gm csf, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
2 mercaptoethanol, by Merck Group (2 mentions)
Ack lysis buffer, by Merck Group (1 mentions)
Opti mem media, by Thermo Fisher Scientific (1 mentions)
Ammonium chloride potassium lysis buffer, by Merck Group (1 mentions)
Opti mem medium, by Thermo Fisher Scientific (1 mentions)
2 protocols using human gp10025 33 hgp100 peptide
1
Generating Bone Marrow-Derived Dendritic Cells
2
Generation and Characterization of Bone Marrow-Derived Dendritic Cells
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BMDCs were generated in accordance with a modified version of a method described previously (Helft et al., 2015 (link); Lou et al., 2004 (link)). Briefly, bone marrow cells isolated from the femurs and tibiae of 7–14-wk-old WT, Aim2−/−, Sting−/−, Cxcl10−/−, Aim2−/−Sting−/−, Aim2−/−Ifnar−/−, Aim2−/−Cxcl10−/−, Il1β−/−, and Il-18−/− mice were filtered through a 70-µm nylon strainer, and red blood cells were lysed by ammonium-chloride-potassium lysis buffer (Sigma Aldrich) and cultured in BMDC medium (RPMI-1640 containing 10% FBS, 100 U/ml PS, 2 mM L-glutamine [Gibco], 50 µM 2-mercaptoethanol [Sigma Aldrich], 20 ng/ml GM-CSF [PeproTech], and 10 ng/ml IL-4 [PeproTech]).
The BMDC medium was replaced on days 3 and 6. On day 8, nonadherent cells were harvested, washed two times with PBS, and used for in vitro experiments. DC purity was assessed by flow cytometry to ensure staining for markers CD11c, MHC II, CD11b, and CD86 on BMDCs. For the generation of peptide-pulsed DC vaccine (DC-gp100), nonadherent cells were pulsed for 3 h at 37°C in 5% CO2 with 10 µM of the human gp10025–33 (hgp100) peptide (GenScript) in Opti-MEM medium (Gibco) and washed three times with PBS before their use.
The BMDC medium was replaced on days 3 and 6. On day 8, nonadherent cells were harvested, washed two times with PBS, and used for in vitro experiments. DC purity was assessed by flow cytometry to ensure staining for markers CD11c, MHC II, CD11b, and CD86 on BMDCs. For the generation of peptide-pulsed DC vaccine (DC-gp100), nonadherent cells were pulsed for 3 h at 37°C in 5% CO2 with 10 µM of the human gp10025–33 (hgp100) peptide (GenScript) in Opti-MEM medium (Gibco) and washed three times with PBS before their use.
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