Assessing Antigen Uptake and Immune Cell Responses

Antigen peptide uptake assay for dendritic cells (DCs) using FITC-MUC1 peptide was performed as previously described (27 (link)). For the assay, FITC-MUC1 peptide (Hi Lyte Fluor 488-GVTSAPDTRPAPGSTA-OH) was custom synthesized (AnaSpec Inc., Fremont CA). ELISpot assay was performed using mouse IFN- γ ELISpot kit (cat no: EL485, RnD Systems, Inc., Minneapolis, MN) as per manufacturer’s protocol. CFSE-based proliferation assay was utilized to measure CD8 T cell proliferation upon co-culture with antigen-antibody loaded DCs using established protocols (27 (link)). CD107a-based degranulation assay was performed to determine the functional activity of NK cells, as described previously (28 (link)).

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Markov S.D., Caffrey T.C., O’Connell K.A., Grunkemeyer J.A., Shin S., Hanson R., Patil P.P., Shukla S.K., Gonzalez D., Crawford A.J., Vance K.E., Huang Y., Eberle K.C., Radhakrishnan P., Grandgenett P.M., Singh P.K., Madiyalakan R., Daniels-Wells T.R., Penichet M.L., Nicodemus C.F., Poole J.A., Jaffee E.M., Hollingsworth M.A, & Mehla K. (2021). IgE-based therapeutic combination enhances anti-tumor response in preclinical models of pancreatic cancer. Molecular cancer therapeutics, 20(12), 2457-2468.

Publication 2021

mouse IFN- γ ELISpot kit

Antibody Antigen Assay Cell proliferation Cfse Co culture Dendritic cells Elispot Fitc Mouse ifn γ Muc1 Nk cells Peptide Peptide hi

Corresponding Organization : University of Nebraska Medical Center

Other organizations : University of California, Los Angeles, Sidney Kimmel Cancer Center, Johns Hopkins University

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Variable analysis

independent variables

FITC-MUC1 peptide
Antigen-antibody loaded DCs

dependent variables

Antigen peptide uptake by dendritic cells (DCs)
IFN-γ production by T cells
CD8 T cell proliferation
Functional activity of NK cells (degranulation)

control variables

Previously described protocols for antigen peptide uptake assay (27)
Mouse IFN-γ ELISpot kit (cat no: EL485, RnD Systems, Inc., Minneapolis, MN)
Established protocols for CFSE-based proliferation assay (27)
Previously described protocols for CD107a-based degranulation assay (28)

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