The following mAb were used: CD3 krome orange [clone UCHT1] (Beckmann Coulter, Brea, CA, USA), CD4 phycoerythrin-cyanin 7 (PE-Cy7) [clone SK3] (BD Bioscience, Franklin Lakes, NJ, USA), CD8 pacific blue [clone B9.11] (Beckmann Coulter), CD11c PE [clone S-HCL3] (BD Bioscience), CD14 fluorescein isothiocyanate (FITC) [clone MϕP9] (BD Bioscience), CD19 allophycocyanin [clone HIB19] (BD Bioscience), CD137 allophycocyanin [clone 4B4-1] (BD Bioscience), CD83 PE [clone HB15] (Miltenyi Biotec), HLA-DP PE [clone B7/21] (21 (link)) (Leinco Technologies, Inc. St.Louis, MO, USA), HLA-DR PE [clone L243] (BD Bioscience), murine IgG2A PE [clone S43.10] (Miltenyi Biotec) and murine IgG3 PE [clone A112-3] (BD Bioscience). Flow cytometric determinations were performed on a Gallios™ 10/3 cytometer (Beckman Coulter, Brea, CA, USA), using the Kaluza for Gallios Acquisition software (Version 1.0, Beckman Coulter) and the data were analyzed with Kaluza Analysis Software (Version 1.3, Beckman Coulter).
Gallios 10 3 cytometer
Manufactured by Beckman Coulter
Sourced in United States
The GALLIOS 10/3 cytometer is a flow cytometry instrument designed for cell analysis. It features 10-color detection and 3-laser excitation. The core function of the GALLIOS 10/3 is to perform quantitative and qualitative analysis of cell samples.
Automatically generated - may contain errors
Lab products found in correlation
Kaluza 1.3 flow analysis software, by Beckman Coulter (3 mentions)
Cytofix cytoperm fixation permeabilization solution kit, by BD (2 mentions)
Kaluza for gallios acquisition software, by Beckman Coulter (1 mentions)
Kaluza analysis software, by Beckman Coulter (1 mentions)
Cd197, by Miltenyi Biotec (1 mentions)
123count ebeads, by Thermo Fisher Scientific (1 mentions)
Liberase dh enzyme, by Roche (1 mentions)
4 protocols using gallios 10 3 cytometer
1
Flow Cytometric Immunophenotyping Protocol
2
PBMC Surface Antigen Staining
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For surface staining, PBMCs (1 × 106 cells in 400 μl PBS containing 1% serum and 0.1% NaN3) were incubated with 1 μg antibody in the dark at 4 °C for 30 min and then washed twice. Antibodies used were as follows: CD45, CD19, CD20, CD56, CD16, CD3, CD4, CD45RA, CD45RO, CD197, CD161, CD25 from Miltenyi and CD5, CD8, CD27 from Beckman Coulter (antibody details shows in Additional file 2 : Table S2). Fluorescently labeled cells were acquired on a GALLIOS 10/3 cytometer and analyzed using Kaluza® 1.3 Flow Analysis Software (Beckman Coulter).
3
Multiparametric Flow Cytometry of Breast Cancer Cells
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Suspensions of PBMC or epithelial cells from fresh BC tissues or cell lines were incubated with the manufacturers' suggested dilution of fluorescently-labeled primary monoclonal antibodies (Table S4). Nuclear labeling of FOXP1 was accomplished using fixed and permeabilized cells (pre-labeled for membrane markers) with the BD Cytofix/Cytoperm™ Fixation/Permeabilization Solution Kit (BD 554714) following the manufacturer's protocol. Absolute cell count beads (123count eBeads; eBioscience) were used to quantify cellular subpopulations. Fluorescently-labeled cell acquired on a GALLIOS 10/3 cytometer and analyzed using Kaluza® 1.3 Flow Analysis Software (Beckman Coulter). Epithelial cell suspensions were prepared from fresh BC tissues by enzyme digestion using optimized conditions (detailed in Supplementary Methods) of Liberase DH enzyme (Roche) and sorted on a Moflo ASTRIOS EQ. 12/4 sorter with gating on EpCAM+ cells.
4
Profiling Tumor-Infiltrating Lymphocytes in Breast Cancer
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TIL in fresh breast tissues from untreated primary BC were analyzed by FACS as previously published (39 (link)). Multi-color FACS was performed using antibodies to well-defined immune cell markers, including CD3, CD4, CD8, CD19 and CD45 and a panel of immune checkpoint molecules: PD-1, PD-L1, PD-L2, CTLA-4, LAG3, and TIM3 (details of the antibodies are provided in Table S2A in Supplementary Material). Intracellular CTLA-4 (iCTLA-4) was labeled by fixing and permeabilizing membrane-labeled cells with the BD Cytofix/Cytoperm™ Fixation/Permeabilization Solution Kit (BD 554714) following the manufacturer’s instructions. Sample data were acquired on a GALLIOS 10/3 cytometer and analyzed using Kaluza® 1.3 Flow Analysis Software (Beckman Coulter, Brea, CA, USA). N = X in the data plots indicate the number of analyzable tumors for a given TIL marker (tumors with insufficient FACS data acquisition were excluded). The amount of material for experimental analysis was limited by the routine requirements of the pathology laboratory, which explains why it was not always possible to analyze all markers for each sample.
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