At different time points after the immunization, blood was collected via mouse tail vein, and the red blood cells were lysed using a RBC lysis buffer solution (eBioscience, San Diego, CA). The monocytes were incubated with Fc block (CD16/32, BioLegend, diluted in FACS buffer at 1:9) at 4 °C for 15 min. The monocytes were then incubated for 30 min at room temperature with PE conjugates of MHC tetramers specific for either CD4 T cells recognizing the OVA epitope ISQAVHAAHAEINEAGR (MBL International, Woburn, MA), CD 8 T cells recognizing the OVA epitope SIINFEKL (MBL International), CD 8 T cells recognizing the gp 100 epitope EGSRNQDWL (MBL International), or CD 8 T cells recognizing the TRP2 epitope SVYDFFVWL (MBL International). Subsequently, the cells were incubated for 10 min at room temperature with a propidium iodide staining solution (eBioscience, diluted in FACS buffer 1:25), CD4-e450 (eBioscience, diluted in FACS buffer 1:200), and CD8α-APC (eBioscience, diluted in FACS buffer 1:200). The cells were washed with FACS buffer, and data were collected on a BD LSR II.
Cd8α apc
Manufactured by Thermo Fisher Scientific
CD8α-APC is a fluorescently labeled antibody that binds to the CD8α surface marker. It is used in flow cytometry applications to identify and quantify CD8+ T cells within a cell population.
Automatically generated - may contain errors
Lab products found in correlation
Cd4 e450, by Thermo Fisher Scientific (1 mentions)
Rbc lysis buffer solution, by Thermo Fisher Scientific (1 mentions)
Propidium iodide staining solution, by Thermo Fisher Scientific (1 mentions)
Cd16 32 fc block, by BioLegend (1 mentions)
Siinfekl, by MBL Life Science (1 mentions)
Cd24 fitc, by Thermo Fisher Scientific (1 mentions)
Cd25 pe, by Thermo Fisher Scientific (1 mentions)
Cd4 apc efluor 780, by Thermo Fisher Scientific (1 mentions)
Cd45.2 apc cy7, by Thermo Fisher Scientific (1 mentions)
Cd45 1 percp cy5, by Thermo Fisher Scientific (1 mentions)
Cd19 percp cy5, by Thermo Fisher Scientific (1 mentions)
Cd69 pe cy7, by Thermo Fisher Scientific (1 mentions)
Cd27 apc, by Thermo Fisher Scientific (1 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
Facscanto 2, by BD (1 mentions)
Facsaria 2, by BD (1 mentions)
Cd86 pe, by Thermo Fisher Scientific (1 mentions)
Apc cd80, by Thermo Fisher Scientific (1 mentions)
Cd11c fitc, by Thermo Fisher Scientific (1 mentions)
Canto 2, by BD (1 mentions)
3 protocols using cd8α apc
1
Quantification of Antigen-Specific T Cells
2
Flow Cytometry Analysis of Immune Cell Subsets
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Cells were analyzed on FACSCantoII (BD) and FlowJo software (Tree Star), and sorted on FACS Aria II (BD). Antibodies used were CD122 PerCP-eFluor 710 (TM-b1), CD19-PerCP-Cy5.5 (eBio1D3), CD24-FITC (M1/69), CD25-PE (PC61.5), CD27-APC (LG.7F9), CD3ε-V500 (500A2), CD4-APC-eFluor 780 (RM4-5), CD44-FITC/APC/APC-eFluor 780 (IM7), CD45.1-PerCP-Cy5.5 (A20), CD45.2-APC-Cy7 (104), NK1.1-PE-Cy7/PerCP-Cy5.5 (PK136), CD62L-FITC/APC/PE-Cy7 (MEL-14), CD69-PE-Cy7 (H1.2F3), CD8α-APC (53–6.7), βTCR-APC-eFluor 780 (H57-597), Qa-2 FITC (69H1-9-9), and γδTCR PerCP-eFluor 710 (eBioGL3; all from eBioscience). iNKT cells were stained at room temperature using α-GalCer–loaded CD1d tetramers. Stainings for intracellular antigens were performed using the FoxP3/Transcription Factor staining buffer set (eBioscience). Active caspase-3+ iNKT cells were stained using PhiPhiLux G1D2 (Oncoimmunin) according to the manufacturer’s recommendations. Subsets are defined as DN1 (CD25−CD44+); DN2 (CD25+CD44+); DN3 (CD25+CD44−); DN4 (CD25−CD44−); ISP (CD25−CD44−CD3ε−CD4−CD8+); DP (CD4+CD8+); ST1 (CD44−NK1.1−); ST2 (CD44hiNK1.1−); ST3 (CD44hiNK1.1+); CD4naive (CD4+CD62LhiCD44−); CD4memory (CD4+CD62L−CD44hi); iNKT (TCRβ+CD1d-αGC+); NKT1a (CD27+IL-2Rβ+); NKT1b (CD27−IL-2Rβ+); NKT2 (CD27+IL-2Rβ−); NKT17 (CD27−IL-2Rβ−); and γδ (γδTCR+).
3
Quantification of Antigen-Specific T Cells
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Analyses of labeled cell suspensions were performed on a custom Canto II (BD) and analyzed with FlowJo software (Tree Star, Inc.). Fluorochrome-conjugated mAbs used were CD11c—FITC (eBioscience, clone N418), CD86—PE (eBioscience, clone GL1), SIINFEKL-H-2Kb—PE (eBioscience, clone 25-D1.16) CD80—APC (eBioscience, clone 16-10A1), CD8α -APC (eBioscience, clone 53–6.7) and I-A/I-E—eFluor450 (MHC II; eBioscience, clone M5/114.15.2). The corresponding isotype controls were also purchased from eBioscience. A PE—dextran-conjugated multimer of SIINFEKL/H-2Kb was purchased from Immudex (Copenhagen, Denmark).
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