End stage of mouse tumors were obtained from BRAFV600E PTEN+/− ATRmD/mD, or BRAFV600E PTEN+/− ATR+/+. Tumors were surgically excised, washed with 70% ethanol and PBS. Tumors were minced with a scalpel and dissociated using a tumor dissociation kit (#130-096-730) and gentleMACs (Miltenyi Biotec, Auburn, CA) according to the manufacture’s protocol. Dissociated tumor cells were labeled by CD3 (17A2)-PE/Cy7, CD19(6D5)-APC and F4/80 (BM8)-APC/Cy7 (BioLegend, San Diego, CA). Cells were subjected to flow cytometry using an Attune Acoustic Focusing Cytometer (Life technology, Grand Island, NY) to analyze the CD3, CD19 and F4/80 positive cells. The resulting data were analyzed using Acoustic Focusing Cytometer software (Life technology, Grand Island, NY, USA).
Cd3 17a2 pe cy7
Manufactured by BioLegend
CD3 (17A2)-PE/Cy7 is a fluorescently labeled antibody that binds to the CD3 protein expressed on the surface of T cells. It can be used to identify and quantify T cells in flow cytometry applications.
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2 protocols using cd3 17a2 pe cy7
1
Isolation and Flow Cytometry of Mouse Tumor Cells
2
Multiparametric flow cytometry of CNS immune cells
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The following fluorochrome-labeled antibodies were used: CD45 (30-11 F) BV510 or FITC 1:100, CD45R/B220 (RA3-6B2) PerCP-Cy5.5 1:100, CD3 (17A2) PE-Cy7 1:200, F4/80 (BM8) APC 1:200, Ly-6G/Ly-6C (RB6-8C5) BV421 1:200, CD11c (N418) AF700 1:150, CD11b (M1/70) PE 1:800 all obtained from Biolegend and NK-1.1 (PK136) APC-Vio770 1:200 obtained from Miltenyi Biotec. After staining, cells were washed twice and resuspended in PBS with 2% FCS. Cells were acquired on a Gallios flow cytometer (Beckman Coulter) or sorted on a FACS Aria III (BD). Sorting was performed using an 85 µm nozzle and 4-way purity sort precision mode. Data were analyzed using FlowJo software v10.6.1 (BD). Cells extracted from the CNS, pia, and dura were sorted for CD45+CD45iv− cells (Supplementary Fig. 2 ). Cell concentrations from all tissues were manually counted in a Fuchs-Rosenthal counting chamber. Sorted single-cell suspensions were used for subsequent single-cell RNA-sequencing.
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