BD Flow Cytometry Reagents Multicolor Panels Designed for Optimal Resolution with the BD LSRFortessa X-20 Cell Analyzer
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1 Multicolor Panels Designed for Optimal Resolution with the BD LSRFortessa X-2 Cell Analyzer Proper multicolor panel design takes into account fluorochrome brightness, antigen density, co-expression, and spillover in the context of a given instrument configuration. The following experiments were designed to maximize resolution of a variety of cell types on a BD LSRFortessa X-2 cell analyzer. BD LSRFortessa X-2 Configuration Laser Fluorochrome Filter BD Horizon BUV /28 Ultraviolet BD Horizon BUV /3 (355 nm) BD Horizon BUV661 67/25 BD Horizon BUV737 74/35 BD Horizon BV421 45/5 BD Horizon BV51 525/5 Violet BD Horizon BV65 61/2 (45 nm) BD Horizon BV65 67/3 BD Horizon BV711 71/5 BD Horizon BV786 78/6 Blue BD Horizon BB515 FITC Alexa Fluor /3 (488 nm) BD Horizon Fixable Viability Stain 52 PerCP-Cy /3 Yellow-Green (561 nm) Red (64 nm) PE 586/15 BD Horizon PE-CF594 61/2 PE-Cy 7 78/6 APC Alexa Fluor /3 BD Horizon APC-R7 Alexa Fluor 7 73/45 APC-H7 APC-Cy7 78/6 Similar configurations exist on the following instruments: BD LSRFortessa cell analyzer, BD FACSAria Fusion cell sorter, and BD FACSAria III cell sorter.
2 Multicolor Panels Designed for Optimal Resolution with the BD LSRFortessa X-2 Cell Analyzer Page 2 B-Cell Panel CD19 HIB19 BD Horizon BUV496 B cells IgM G2-127 PE B-cell subset IgD IA6-2 BD Horizon BUV395 B-cell subset CD24 ML5 BD Horizon BV51 /naïve subset M-T271 BD Horizon BV421 subset CD2 2H7 APC-H7 Plasmablast subset CD38 HIT2 PerCP-Cy 5.5 Plasmablast subset 5514 Viability n/a BD Horizon Fixable Viability Stain 52 Cellular viability PBMCs were prepared by using Ficoll-Paque Plus according to the manufacturer s directions and incubated with antibodies in the presence of BD Horizon Brilliant Stain Buffer at room temperature protected from light for 2 minutes and washed Analysis of Human B-Cell Subsets B Cells Switched Switched Unswitched Naive CD Plasmablasts IgD CD2 B Cells FVS B Cells CD Transitional CD CD38 B Cells -18 IgD IgM
3 Multicolor Panels Designed for Optimal Resolution with the BD LSRFortessa X-2 Cell Analyzer Page 3 T-Cell Panel CD3 UCHT1 BD Horizon BB515 T cells CD4 SK3 BD Horizon BUV395 Helper T-cell subset CD8 SK1 APC-H7 Cytotoxic T-cell subset CD127 HIL-7R-M21 BD Horizon BV786 Treg identification CD25 M-A251 PE Treg identification M-T271 BD Horizon BV421 Antigen experience HI1 APC Antigen experience /CCR BD Horizon PE-CF594 Antigen experience Brilliant Stain Buffer at room temperature and protected from light for 2 minutes. Analysis of Human T-Cell Populations Terminal Central CD8-1, CD3 + T Cells CD Late Central 1 2 Central Treg CD Treg Treg CD
4 Multicolor Panels Designed for Optimal Resolution with the BD LSRFortessa X-2 Cell Analyzer Page 4,, and T-Cell Panel CD3 UCHT1 PerCP-Cy5.5 T cells CD4 SK3 BD Horizon BUV496 CD4+ subset CD8 SK1 APC-H7 CD8+ subset M-T271 BD Horizon BV421 Antigen experience APC Antigen experience HI1 FITC Antigen experience CD45RO UCHL1 BD Horizon BUV395 Antigen experience /CCR BD Horizon PE-CF594 Antigen experience Brilliant Stain Buffer at room temperature and protected from light for 2 minutes. Analysis of Human,, and T-Cell Populations (CD8 + ) CD3 + T Cells CD45RO CD , CD / Central / Central Late Early Central
5 Multicolor Panels Designed for Optimal Resolution with the BD LSRFortessa X-2 Cell Analyzer Page 5 Analysis of Human,, and T Cell Populations (CD4 + ) CD3 + T Cells CD CD45RO CD RTE Central Central Th1 Th1/Th2 / Central
6 Multicolor Panels Designed for Optimal Resolution with the BD LSRFortessa X-2 Cell Analyzer Page 6 Regulatory T-Cell (Treg) Panel CD3 UCHT1 BD Horizon BV711 T cells CD4 SK3 BD Horizon BUV395 Helper T-cell subset CD25 M-A251 BD Horizon PE-CF594 Treg identification CD127 HIL-7R-M21 Alexa Fluor 647 Treg identification G46-6 PE-Cy7 Treg subset CD194/CCR4 1G1 BD Horizon BV421 Antigen experience CD45RO UCHL1 BD Horizon BB515 Antigen experience Brilliant Stain Buffer at room temperature and protected from light for 2 minutes. Analysis of Human Treg Populations CD CD Treg CD CD25 CD Treg Treg Treg CD45RO and Treg Early Contact-Dependent Treg CD45RO
7 BD Flow Cytometry Reagents Multicolor Panels Designed for Optimal Resolution with the BD LSRFortessa X-2 Cell Analyzer Page 7 Dendritic Cell Panel CD3 SK7 BD Horizon BV786 Exclusion (T cell) CD14 M5E2 BD Horizon BV786 Exclusion (monocyte) CD19 SJ25C1 BD Horizon BV786 Exclusion (B cell) CD56 NCAM16.2 BD Horizon BV786 Exclusion (granulocyte) G46.6 BD Horizon BV421 Dendritic cells (DC) CD11c B-ly6 BD Horizon BB515 Myeloid DC (mdc) Clec9a 3A4/Clec9A PE mdc Subset CD141 1A4 APC mdc Subset CD123 7G3 BD Horizon BUV395 Plasmacytoid DC (pdc) Brilliant Stain Buffer at room temperature and protected from light for 2 minutes. Analysis of Human Dendritic Cell Populations DC Scatter Gate (x1,) SSC DC Scatter Gate Lineage Negative + DCs FSC CD3/CD14/CD19/CD56 Lineage Negative + DCs Myeloid DC CD Plasmacytoid DC Myeloid DC CD Clec9a mdc Clec9a + mdc CD11c Clec9A
8 BD Flow Cytometry Reagents Multicolor Panels Designed for Optimal Resolution with the BD LSRFortessa X-2 Cell Analyzer Page 8 Myeloid Cell Panel CD3 UCHT1 BD Horizon BV51 Exclusion (T cell) CD19 SJ25C1 BD Horizon BV51 Exclusion (B cell) CD2 2H7 BD Horizon BV51 Exclusion (B cell) G46-6 FITC DC, NK, monocyte subsets CD16 3G8 BD Horizon BUV737 NK cell/monocyte subset CD56 NCAM 16.2 BD Horizon BV421 NK cells CD11c B-Ly6 BD Horizon PE-CF594 DC subset CD123 7G3 BD Horizon BUV395 pdc subset CD14 M5E2 APC Monocytes CD Monocytes Granulocyte/DC Brilliant Stain Buffer at room temperature and protected from light for 2 minutes. Analysis of Myeloid Cells (Dendritic Cells (DC) and Monocytes) SSC (x1,) Granulocyte/DC Granulocytes Dendritic Cells CD Plasmacytoid DC Dendritic Cells Myeloid DC , CD3/CD19/CD CD11c CD Classical Monocytes Non-classical Analysis of Human Natural Killer (NK) Cells Lineage Neg Mature and Immature NK Cells CD Lineage Neg CD Immature NK Cells Mature NK Cells CD Activated NK Cells CD3/CD19/CD2 Class 1 Laser Product. Research Use Only. Not for use in diagnostic or therapeutic procedures. APC-Cy7: US patent 5,714,386 Alexa Fluor is a registered trademarks of Life Technologies Corporation. CF is a trademark of Biotium, Inc. Ficoll-Paque is a trademark of GE Healthcare. Cy is a trademark of GE Healthcare. Cy dyes are subject to proprietary rights of GE Healthcare and Carnegie Mellon University, and are made and sold under license from GE Healthcare only for research and in vitro diagnostic use. Any other use requires a commercial sublicense from GE Healthcare, 8 Centennial Avenue, Piscataway, NJ , USA. BD, BD Logo and all other trademarks are property of Becton, Dickinson and Company. 215 BD CD
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