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Monoclonal Antibodies

Anti-gp100 T-cell Hybridoma [BUSA14]

Invented by Lea Eisenbach
Invented at Weizmann Institute Of Science
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BUSA14 cells are efficiently activated by mouse and human gp10025–33 peptides. DC2.4 cells were loaded with hgp10025–33 at doses ranging from 0.5 to 10000 ng/ml (A) or mgp10025–33 (10 to 50000 ng/ml)(B). Then co-cultured with BUSA14 for 12 hours. Cells were then lysed and β-Gal enzymatic activity was detected with CPRG. Representative results (1 of 3 experiments) are presented as ΔOD (sample OD-background OD) measured after 3 hours. C. Sixty thousand BUSA14 cells/well were incubated in triplicate overnight with serial dilutions of DC2.4 cells loaded with 50 µg/ml hgp10025–33 or mgp10025–33. SIINFEKL loaded DC2.4 cells were used as negative control. Assays were done as before and presented as ΔOD measured after 24 hours
T cell markers expressed by BUSA14 cells. A. BUSA14, B3Z and BWZ.36/CD8α cells were analyzed by flow cytometry and monoclonal antibodies against CD8 and TCR Vβ13 to confirm the Pmel-1 TCR expression. B. BUSA14, BWZ.36/CD8α and B3Z cells were co-cultured with hgp10025–33 or SIINFEKL for 12 hours, or remained untreated. Cells were stained with antibodies to CD8, CD62L and CD44 and analyzed by flow cytometry. Cells were gated for CD8 to exclude DC2.4 cells. C. Two million BUSA14, BWZ.36/CD8α and B3Z cells were co-cultured with 6×105 DC2.4 loaded with hgp10025–33 or SIINFEKL for 12 hours, or remained untreated. Then stained with antibodies against CD8, CD69, CD279 and analyzed by flow cytometry. Cells were gated for CD8 to exclude DC2.4 cells. Mean fluorescent intensity (MFI) values are presented in the figure. This figure is a representative of three experimental repeats.
Detection of cytokines produced by BUSA14 cells. BUSA14 or BWZ.36/CD8α were co-incubated with DC2.4 cells loaded with hgp10025–33 or SIINFEKL. Cells alone or co-cultured with unloaded DC2.4 or with PMA and ionomycin served as negative and positive controls, respectively. All cells were intracellulary stained with antibodies to CD8, IL-2/TNFα (A), IL-4/IFNγ (B) and analyzed by flow cytometry. Cells were gated for CD8 to exclude DC2.4 cells.
BUSA14 are activated by hgp10025–33 and mgp10025–33 presented on melanoma cell lines. A. B16-MO5, F10.9, D122 and EL4 tumor cell lines were analyzed by flow cytometry with monoclonal antibodies to H-2Kb and H-2Db to analyze MHC-I membranal expression. MFI values are presented in the figure. B. Twenty thousand B16-MO5, F10.9 and D122 cells were loaded with 30 µg/ml hgp10025–33 or SIINFEKL peptides. Cells were washed and co-incubated with 6×104 BUSA14 and BWZ.36/CD8α for 12 hours. Cells were then lysed and β-Gal enzymatic activity was monitored with CPRG. Cultures with D122 served as reference for CPRG background levels. Representative results (1 of 3 experiments) are presented as ΔOD (sample OD-background OD) measured after 12 hours. C. Sixty thousand BUSA14 and BWZ.36/CD8α cells/well were incubated overnight, in triplicates, with 2×104 B16-MO5, F10.9 or D122 tumor cell lines. Representative results (1 of 2 experiments) are presented as ΔOD (sample OD-background OD) measured after 24 hours. Statistical analysis was done using student T test (*p<0.05, **p<0.01, ***p<0.001).
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Catalogue Number 160883
Applications N/A
Antigen/Gene or Protein Targets gp100(25-33) melanoma antigens
Reactivity N/A
Relevance This hybridoma expresses a variety of membranal T cell markers and secretes IL-2 and TNFα. The activated lacZ hydrolyzes the yellow chlorophenol red beta-D galactopyranoside (CRPG) to a dark red color, enabling an easy assay. Thus, BUSA14 offers a quantifiable, cheap and straightforward tool for studying peptide presentation by MHC-I molecules on the cell surface.
Host Mouse
Recommended Growing Conditions RPMI1640, 10%FCS, 2mM glutamine, 1% sodium pyruvate, 1% non-essential amino acids, 5X10 (-5) beta mercapto ethanol, 1% antibiotics.
Notes Hybridoma cell line [clone BUSA14] which generates lacZ inducible CD8+ T -cells capable of recognising both human and mouse gp100(25-33) melanoma antigens presented on dendritic and tumour cell lines.
Conditional: inducible LacZ
Total splenocytes were isolated from spleens of Pmel-1 mice and grown in lymphocyte medium. As sensitizing cells, Two ml were incubated with 30 µg/ml hgp10025–33 peptide for 2 hours, diluted in 10 ml lymphocyte medium and added to the flasks. Four days later, Cells were washed once with PBS, separated on Lympholyte-M and fused with the BWZ.36/CD8α cells using polyethylene glycol.
Research Area Cancer, Immunology

References

There are 1 reference entries for this reagent.

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References: 1 entry

Cafri et al. 2013. PLoS One. 8(2):e55583. PMID: 23383339.

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References: 1 entry

Cafri et al. 2013. PLoS One. 8(2):e55583. PMID: 23383339.

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