Isolated CD8+ OT-I cells were labeled with 1 μM ratiomeric calcium-binding dye Indo-1, AM (Life Technologies) in PBS for 15 min at 37 °C. Cells were washed twice with complete RPMI, and incubated at 37 °C for 15 min to allow for complete de-esterification. Cells were coated with 5 μg/ml anti-CD3 (2C11; UCSF Hybridoma Core) on ice, washed, and transferred to 37 °C 10 min prior to data collection. To induce CD3 cross-linking, 10 μg/ml of anti-Armenian hamster (BioLegend) was added to CD3-coated cells after 1 min of sample collection. Alternatively, 1 μM of thapsigargin (Sigma-Aldrich) was added to uncoated cells. Cell samples were kept in a heating chamber (37 °C) during data collection by flow cytometry.
Anti armenian hamster
Manufactured by BioLegend
Anti-Armenian hamster is a lab equipment product. It is used for the detection and/or quantification of Armenian hamster-derived proteins or other biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Indo 1 am, by Thermo Fisher Scientific (2 mentions)
Thapsigargin, by Merck Group (2 mentions)
Cleaved casp3, by Cell Signaling Technology (1 mentions)
Tcs sp5 2, by Leica (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Las af, by Leica (1 mentions)
Dfc360 fx, by Leica (1 mentions)
F4 80, by Thermo Fisher Scientific (1 mentions)
Dfc420c, by Leica (1 mentions)
Dmi4000b, by Leica (1 mentions)
In situ cell death detection kit, by Roche (1 mentions)
Cytokeratin 19, by Abcam (1 mentions)
Gsdmd, by Abcam (1 mentions)
3 protocols using anti armenian hamster
1
Calcium Signaling in CD8+ T Cells
2
Calcium Signaling in CD8+ T Cells
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Isolated CD8+ OT-I cells were labeled with 1 μM ratiomeric calcium-binding dye Indo-1, AM (Life Technologies) in PBS for 15 min at 37 °C. Cells were washed twice with complete RPMI, and incubated at 37 °C for 15 min to allow for complete de-esterification. Cells were coated with 5 μg/ml anti-CD3 (2C11; UCSF Hybridoma Core) on ice, washed, and transferred to 37 °C 10 min prior to data collection. To induce CD3 cross-linking, 10 μg/ml of anti-Armenian hamster (BioLegend) was added to CD3-coated cells after 1 min of sample collection. Alternatively, 1 μM of thapsigargin (Sigma-Aldrich) was added to uncoated cells. Cell samples were kept in a heating chamber (37 °C) during data collection by flow cytometry.
3
Multimodal Histopathological and Immunofluorescence Analyses
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Histopathological analyses were performed on formalin-fixed paraffin-embedded tissue after Mayer’s haematoxylin and eosin (H&E) staining. Immunofluorescence staining of tissue sections was performed using the Alexa Fluor and TSA Cy3/Fluorescein system as recommended by the manufacturer (Perkin&Elmer). The following antibodies were used: murine MLKL (Biorbyt, Cat.No.: orb32399), GSDMD (abcam, Cat.No.: ab209845), Cytokeratin 19 (abcam, Cat.No.: ab52625), cleaved CASP3 (Cell Signaling, Cat.No.: 9661S), Albumin (abcam, Cat.No.: ab106582), CD11c (BD Bioscience, Cat.No.: 550283), F4/80 (eBioscience, Cat.No.: 14-4801-85), Myeloperoxidase (MPO) (abcam, Cat.No.: ab9535), and secondary anti-rabbit (Dianova, Cat.No.: 111-065-144), anti-chicken (abcam, Cat.No.: ab150169), anti-Armenian hamster (BioLegend, Cat.No.: 405501), anti-rat (Bioscience, Cat.No.: 554014), or anti-rabbit-Alexa 647 (BioLegend, Cat.No.: 406414). Nuclei were counterstained with Hoechst 33342 (Invitrogen). Cell death (TUNEL) was analysed using the in-situ cell death detection kit (Roche). Images were obtained using a confocal fluorescence microscope (LEICA TCS SP5 II) or the microscope LEICA DMI 4000B together with the LEICA DFC360 FX or LEICA DFC420C camera and the imaging software “LAS AF” (Leica). Oil red staining was performed on cryosections as recommended by protocol described before22 .
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