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H 2kb clone 25 d1

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H-2Kb (clone 25-D1.16) is a monoclonal antibody that recognizes the mouse MHC class I molecule H-2Kb. It can be used for the detection and analysis of H-2Kb-expressing cells by flow cytometry.

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Lab products found in correlation

Geneticin, by Thermo Fisher Scientific (1 mentions) Fugene hd, by Promega (1 mentions)

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H-2Kb

4 protocols using h 2kb clone 25 d1

1

Generation of OVA-expressing B16 F10 cells

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B16 F10 (mouse melanoma) cells were purchased from ATCC (CRL-6475). Ovalbumin-expressing B16 F10 (B16 F10 OVA) cells were generated in-house by Dr Pedro Velica by co-transfection of the transposon vector pT2 encoding OVA, eGFP and neomycin phosphotransferase and the vector encoding transposase SB11. After three days, 400 μg/ml G418 (Gibco, 10131035) was added to culture media to select for transgene-expressing cells. Successful integration was confirmed by analysing eGFP fluorescence by flow cytometry. Limiting dilution was used to derive monoclonal OVA-expressing lines for each cell line. OVA presentation was confirmed by flow cytometry using a PE-labelled antibody against surface SIINFEKL bound to H-2Kb (clone 25-D1.16, BioLegend).
Bargiela D., Cunha P.P., Veliça P., Krause L.C., Brice M., Barbieri L., Gojkovic M., Foskolou I.P., Rundqvist H, & Johnson R.S. (2024). The factor inhibiting HIF regulates T cell differentiation and anti-tumour efficacy. Frontiers in Immunology, 15, 1293723.
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2

Generating OVA-expressing Tumor Cells

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B16-F10, LLC and cells were co-transfected with the transposon vector pT2 encoding OVA, eGFP and neomycin phosphotransferase and the vector encoding transposase SB11. Three days later 400 mg/ml G418 (Gibco, 10131035) was added to culture media to select for transgene-expressing cells. Successful integration was confirmed by analyzing eGFP fluorescence by flow cytometry. Limiting dilution was used to derive monoclonal OVA-expressing lines for each cell line. OVA presentation was confirmed by flow cytometry using a PE-labeled antibody against surface SIINFEKL bound to H-2Kb (clone 25-D1.16, BioLegend).
Rundqvist H., Veliça P., Barbieri L., Gameiro P.A., Bargiela D., Gojkovic M., Mijwel S., Reitzner S.M., Wulliman D., Ahlstedt E., Ule J., Östman A, & Johnson R.S. (2020). Cytotoxic T-cells mediate exercise-induced reductions in tumor growth. eLife, 9, e59996.
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3

Generating OVA-expressing Monoclonal Cell Lines

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B16-F10, LLC, and MC38 were co-transfected using FugeneHD (Promega, #E2311/2) with the transposon vector pT2 encoding OVA, eGFP and neomycin phosphotransferase, and the vector encoding transposase SB11. Three days later 400 mg/ml geneticin (Thermo Fisher, #10131027) was added to culture media to select for transgene-expressing cells. Successful integration was confirmed by analyzing eGFP fluorescence by flow cytometry. Limiting dilution was used to derive monoclonal OVA-expressing lines for each cell line. OVA presentation was confirmed by flow cytometry using a PE-labeled antibody against surface SIINFEKL bound to H-2Kb (clone 25-D1.16, BioLegend).
Veliça P., Cunha P.P., Vojnovic N., Foskolou I.P., Bargiela D., Gojkovic M., Rundqvist H, & Johnson R.S. (2021). Modified Hypoxia-Inducible Factor Expression in CD8+ T Cells Increases Antitumor Efficacy. Cancer immunology research, 9(4), 401-414.
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4

Generation and Characterization of B16 F10 OVA Cell Line

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B16 F10 (mouse melanoma) cells were purchased from ATCC (CRL-6475). Ovalbumin-expressing B16 F10 (B16 F10 OVA) cells were generated in-house by co-transfection of the transposon vector pT2 encoding OVA, eGFP and neomycin phosphotransferase and the vector encoding transposase SB11. After three days, 400 μg/ml G418 (Gibco, 10131035) was added to culture media to select for transgene-expressing cells. Successful integration was confirmed by analyzing eGFP fluorescence by flow cytometry. Monoclonal OVA-expressing lines were derived by a process of limiting dilution. OVA presentation was confirmed by flow cytometry using a PE-labelled antibody against surface SIINFEKL bound to H-2Kb (clone 25- D1.16, BioLegend).
Bargiela D., Cunha P.P., Veliça P., Foskolou I.P., Barbieri L., Rundqvist H, & Johnson R.S. (2022). Vitamin B6 Metabolism Determines T Cell Anti-Tumor Responses. Frontiers in Immunology, 13, 837669.
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