Egsrnqdwl

Manufactured by AnaSpec

EGSRNQDWL is a laboratory equipment product manufactured by AnaSpec. It serves as a core function in various scientific applications, but a detailed description cannot be provided while maintaining an unbiased and factual approach.

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Lab products found in correlation

3h thymidine, by GE Healthcare (5 mentions) Streptavidin microbeads, by Miltenyi Biotec (3 mentions) Facsaria, by BD (2 mentions) Facsaria cell sorter, by BD (2 mentions) Topcount nxt, by PerkinElmer (1 mentions) Topcount nxt instrument, by PerkinElmer (1 mentions) Siinfekl, by AnaSpec (1 mentions) Svydffvwl, by AnaSpec (1 mentions)

Spelling variants of this product

Mgp10025-33 EGSRNQDWL (2 citations)

6 protocols using egsrnqdwl

Measuring MDSC-Mediated T Cell Suppression

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PMN-MDSCs (Ly6G⁺) were purified from spleens and tumors. Isolated cells were subsequently incubated with biotinylated Ly6G antibody and streptavidin microbeads (Miltenyi). M-MDSCs (CD45⁺CD11b⁺Ly6C^hiLy6G⁻) and macrophages (CD45⁺CD11b⁺F4/80⁺Ly6C⁻) were sorted using FACSAria (BD Biosciences). PMN-MDSCs, M-MDSCs, or macrophages were plated in U-bottom 96-well plates (3 replicates) in RPMI supplemented with 10% FBS, penicillin–streptomycin, and 0.05 mmol/L 2-mercaptoethanol, and cocultured at different ratios with splenocytes from PMEL mice in the presence of 0.1 μg/mL of murine gp100 peptide (EGSRNQDWL, AnaSpec, Inc.). After 48 hours, the cells were incubated with ³H-thymidine (1 μCi/well; GE Healthcare) for 16 hours. Proliferation was measured using the TopCount NXT instrument (PerkinElmer).

Hashimoto A., Sarker D., Reebye V., Jarvis S., Sodergren M.H., Kossenkov A., Sanseviero E., Raulf N., Vasara J., Andrikakou P., Meyer T., Huang K.W., Plummer R., Chee C.E., Spalding D., Pai M., Khan S., Pinato D.J., Sharma R., Basu B., Palmer D., Ma Y.T., Evans J., Habib R., Martirosyan A., Elasri N., Reynaud A., Rossi J.J., Cobbold M., Habib N.A, & Gabrilovich D.I. (2021). Upregulation of C/EBPα Inhibits Suppressive Activity of Myeloid Cells and Potentiates Antitumor Response in Mice and Patients with Cancer. Clinical Cancer Research, 27(21), 5961-5978.

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Suppression of PMEL-specific CD8+ T cell proliferation by MDSC and TAM

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PMN-MDSCs (Ly6G⁺) were purified from spleens and tumors by subsequently reaction with biotinylated Ly6G antibody and streptavidin microbeads (Miltenyi), and pass through the MACS column according to the manufacturer’s recommendation (Miltenyi). The purity of cell populations was >95%. M-MDSC (CD45+, CD11b+, Ly6G−, Ly6C+) cells and TAM (CD45+, CD11b+, F4/80+, Ly6C−, Ly6G−) were isolated from spleens (M-MDSC) and tumors by cell sorting on FACSAria cell sorter (BD Biosciences). CD8⁺ T cells from PMEL mice that recognize gp100-derived peptide were used as responders. Spleen cells from PMEL mice were suspended in complete RPMI media and plated into 96-well U-bottom plates at 10⁵ cells/well. PMN-MDSC, M-MDSC or TAM were added to the wells at 12,500 to 10⁵ cells/well (1:8-1:1 ratios). Murine gp100 peptide (25 (link)-33) EGSRNQDWL (AnaSpec, Inc.) was added into the wells at the final concentration of 0.1 μg/mL. After 48 hours of culture, cells were pulsed with ³H-thymidine (1 μCi/well; GE healthcare) for 16 hours. ³H-thymidine uptake was counted using a liquid scintillation counter as counts per minute (cpm) and calculated the percentage of proliferation to the positive control (responder cells and peptide without MDSC or TAM).

Hashimoto A., Fukumoto T., Zhang R, & Gabrilovich D. (2020). Selective targeting of different populations of myeloid-derived suppressor cells by histone deacetylase inhibitors. Cancer immunology, immunotherapy : CII, 69(9), 1929-1936.

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MDSC Isolation and Suppression Assay

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PMN-MDSCs (Ly6G⁺) were purified from spleens and tumors. Isolated cells were subsequently incubated with biotinylated Ly6G antibody and streptavidin microbeads (Miltenyi). M-MDSCs (CD45⁺CD11b⁺Ly6C^hiLy6G⁻) and macrophage (CD45⁺CD11b⁺F4/80⁺Ly6C⁻) were sorted using FACS Aria (BD Biosciences). PMN-MDSC, M-MDSC or macrophages were plated in U-bottom 96-well plates (3 replicates) in RPMI supplemented with 10% FBS, Penicillin-Streptomycin and 0.05 mM 2-mercaptoethanol, and co-cultured at different ratios with splenocytes from PMEL mice in the presence of 0.1 μg/mL of murine gp100 peptide (EGSRNQDWL, AnaSpec, Inc.). After 48 h, the cells were incubated with ³H-thymidine (1 μCi/well; GE healthcare) for 16 h. Proliferation was measured using the TopCount NXT instrument (PerkinElmer).

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Suppression of CD8+ T Cell Proliferation by Myeloid Cells

Cited 1 time

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Ly6G⁺ cells were purified from spleen cells or tumor cells by positive selection using biotinylated Ly6G antibody and streptavidin microbeads (Miltenyi). The purity of the cell populations was >95%. CD11b⁺Ly6C^highLy6G⁻ cells were isolated from spleen cells by cell sorting on a FACSAria cell sorter (BD Biosciences). CD8⁺ T cells from PMEL mice that recognize the gp100-derived peptide, were used as responders. Splenocytes from PMEL mice were mixed with splenocytes from naïve mice at 1:4 ratio in complete RPMI media and plated into 96-well U-bottom plates at 10⁵ cells/well. Ly6G⁺ or CD11b⁺Ly6C^highLy6G⁻ cells were added to the wells at 1:16–1:1 ratios. Murine gp100 peptide (25 (link)–33 (link)) EGSRNQDWL (AnaSpec, Inc.) was added into the wells at the final concentration of 0.1 μg/mL. After 48 hours, cells were pulsed with ³H-thymidine (1 μCi/well; GE healthcare) for 16 hours. ³H-thymidine uptake was counted using a liquid scintillation counter as counts per minute (cpm) and calculated the percentage of proliferation to the positive control (the wells with responder cells and peptide but without suppressive cells).

Hashimoto A., Gao C., Mastio J., Kossenkov A., Abrams S.I., Purandare A.V., Desilva H., Wee S., Hunt J., Jure-Kunkel M, & Gabrilovich D.I. (2018). Inhibition of casein kinase 2 disrupts differentiation of myeloid cells in cancer and enhances the efficacy of immunotherapy in mice. Cancer research, 78(19), 5644-5655.

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MDSC-Mediated Suppression of T Cell Proliferation

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MDSCs were purified from the spleens, bone marrow, or tumors. The purity of cell populations was >95%. The PMEL-1 and OT-I responder mice have CD8⁺ T cells which recognize gp100-derived and OVA-derived peptides, respectively. Splenocytes from PMEL-1 or OT-I mice were mixed with splenocytes from naïve mice at the 1:4 ratio in the complete RPMI media, and then plated into 96-well U-bottom plates at 10⁵ cells per well. CD11b⁺/Ly6G⁺ or CD11b⁺/Ly6C⁺ MDSCs were added to the wells at 0.25, 0.5 or 1 × 10⁵ cells per well. The murine gp100 peptide (amino acids 25~33), EGSRNQDWL (AnaSpec), or OVA peptide (amino acids 257~264), SIINFEKL (AnaSpec) was dissolved in pure water, diluted with the RPMI complete media, and then added into the wells at the final concentration of 0.1 μg/mL. After incubation for 48 h, cells were radiolabeled with ³H-thymidine (1 μCi per well; GE Healthcare) for 6 h. The uptake of ³H-thymidine was measured as counts per minute (CPM) using a liquid scintillation counter. The percentage of proliferation in comparison to positive controls (the wells with responder cells and the corresponding peptide) was calculated.

Tang C.H., Chang S., Hashimoto A., Chen Y.J., Kang C.W., Mato A.R., Del Valle J.R., Gabrilovich D.I, & Hu C.C. (2018). Secretory IgM exacerbates tumor progression by inducing accumulations of MDSCs in mice. Cancer immunology research, 6(6), 696-710.

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Melanoma Vaccine with TLR3 Agonist

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Melanoma vaccine was freshly prepared prior to inoculation. Vaccine consisted of 10 μg each peptide premelanosome protein (Pmel17 or gp100) (25-30; seq: EGSRNQDWL, Anaspec) and tyrosinase-related protein-2 (TRP-2) (180-188; seq: SVYDFFVWL, Anaspec), and 50 μg Poly I:C (TLR3 agonists). To deplete IL-10, some mice were given three i.p. injections of anti-IL-10 (JES5-2A5 clone, BioXcell) at days 2 (300μg), 5 (100μg) and 22 (100μg).

Tewari A., Prabagar M.G., Gibbings S.L., Rawat K, & Jakubzick C.V. (2021). LN Monocytes Limit DC-Poly I:C Induced Cytotoxic T Cell Response via IL-10 and Induction of Suppressor CD4 T Cells. Frontiers in Immunology, 12, 763379.

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