Muse Assays for Cell Analysis

Muse Assays for Cell Analysis

Multiple Assay Outputs for Cell Analysis Cell Health Cell Signalling Immunology Muse Count & Viability Kit Muse Cell Cycle Kit Muse Annexin V & Dead Cell Kit Muse Caspase 3,7 Kit Muse MultiCaspase Kit Muse MitoPotential Kit Muse Nitric Oxide Kit Muse Oxidative Stress Kit Muse Ki67 Proliferation Kit Muse LC3 Autophagy (Ab Based) Muse LC3 Autophagy (Reporter Cell Line) Muse H2A.X Activation Dual Detection Kit Muse MAPK Activation Dual Detection Kit Muse EGFR-RTK Activation Dual Detection Kit Muse PI3 Activation Dual Detection Kit Muse Bcl-2 Activation Dual Detection Kit Muse Multi-Color DNA Damage Kit Muse PI3K/MAPK Activation Dual Detection Kit Muse Human CD4 T Cell Kit Muse Human CD8 T Cell Kit Muse Human B Cell Kit Muse Human CD25 Lymphocyte Kit Muse Human CD69 Lymphocyte Kit Nucleic Acid Muse SmartFlare Live Cell RNA Detection Kit 2

Cell Health Assays

Muse Count and Viability Assay MCH100102 100 Tests MCH600103 600 Tests MCH100104 for difficult samples Uses 2 dyes to assess cellular viability Viable cells have intact membranes and are nonpermeable to charged dyes Dead cells have permeability to charged dyes Inclusion of a nucleated cell dye allows identification of all cells over debris or non-nucleated cells Muse Assay Features/Benefits: Total prep time <15 min (simple Mix-and-Read assay) OK for wide variety of cell types (adherent and suspension cell lines) Wide concentration range of sample OK Minimal sample volume required Non-Viable Cell Not a Cell Viable Cell Kit detects: Concentrations of viable cells, dead cells and total cells. % viability of cells Distinction between late apoptotic and live cells Muse Count and Viability Reagent 4

Muse Annexin V & Dead Cell Assay (MCH100105) Determines number and percentage of cells undergoing apoptosis (early apoptotic cells) by the binding of Annexin V-PE to phosphatidylserine, a membrane component translocated to the cell surface during apoptosis. Determines number of late apoptotic and dead cells based on loss of membrane integrity Inclusion of 7AAD in the assay allows for the detection of dead/late apoptotic cells with permeabilized membranes. Muse Annexin V & Dead Cell Reagent 5

Muse Cell Cycle Assay (MCH100106) Kit detects percentage of cells in various stages of cell cycle Propidium iodide intercalates into the base pairs of dsdna Fluorescent intensity of a stained cell is directly proportional to the DNA content of the cell, and consequently, reflects phase of cell cycle Muse Cell Cycle Reagent 6

Muse Caspase-3/7 Assay (MCH100108) Kit detects percentage and concentration of cells in various stages of apoptosis, based on Caspase-3/7 activity, in combination with a dead cell dye. Kit contains Caspase 3/7 Reagent which contains a DNA binding dye linked to a Caspase 3/7 substrate. When bound to DEVD substrate the dye s ability to electrostatically bind to DNA is inactivated. Cleavage by active caspases results in release of the dye and translocation to the nucleus Upon binding to DNA, dye becomes highly fluorescent Inclusion of 7AAD in the assay allows for the detection of dead/late apoptotic cells with permeabilized membranes. NucView Caspase Substrate 7 7AAD Assay Buffer

Muse Multi-Caspase Assay (MCH100109) Kit detects percentage and concentration of cells in various stages of apoptosis, based on Multiple Caspase activity, in combination with a dead cell dye. Muse Multi-Caspase Assays is a mix and read assay for Pan caspase, that can detect the presence of multiple caspases (Caspase 1,3,4,5,6, 7, 8 & 9) in combination with a dead cell dye. Assay uses a Fluorescent-Labeled Inhibitor of Caspases (FLICA) Binds to caspases which are activated during apoptosis; resulting fluorescent signal is proportional to the number of active caspases in cell. Also detects cells in late apoptosis based on staining with a membrane impermeant DNA intercalator (inclusion of dead cell dye 7AAD) MultiCaspase Reagent 7AAD 10 X Assay Buffer + 1X PBS 8 Anhydrous DMSO

Muse MitoPotential Assay (MCH100110) Muse MitoPotential Assay is a simple no-wash assay that provides percentage and concentration of cells demonstrating mitochondrial membrane depolarization and cell death. Assay is based on detection of mitochondrial potential changes using Muse MitoPotential Reagent which is a cationic lipophilic dye. High membrane potential drives accumulation of dye within inner membrane of intact mitochondria resulting in high fluorescence. Depolarized cells are detected by decreased fluorescence. 7AAD enters cells that are compromised in dead or late apoptotic cells. 9 Muse MitoPotential Dye 7AAD 1 X Assay Buffer

Muse Nitric Oxide Kit (MCH100112) Assay Prinicple: Simplified assay on Muse to detect Nitric Oxide (NO) and Nitric Oxide Synthase (NOS) based activity in combination with cell death. Based on DAX-J2 Orange a novel, non-fluorescent dye that is cell permeable and becomes fluorescent product upon NO oxidation. Photostable, & ph independent advantage over other probes. Inclusion of 7AAD in the assay allows for the detection of dead/late apoptotic cells with permeabilized membranes. NO(-) Dead cells Negative (Live) cells RAW 264.7 Cells treated with LPS + IFNɣ and analyzed with Muse Nitric Oxide Assay NO(+) Dead cells NO(+) Live cells Nitric Oxide Detection Reagent 7AAD Reagent 1X Assay Buffer 1X Assay Buffer BA 10

Muse Oxidative Stress Kit (MCH100111) Simplified assay on Muse for the detection of ROS (reactive oxygen species) in cells, esp superoxide radicals. Based on Dihydroethidium, a well characterized and cell permeable reagent which exhibits blue fluorescence in cytosol, but red fluorescence when oxidized and bound to DNA. Simultaneously determines the count and percentage of cells undergoing oxidative stress Negative cells ROS(+) cells Optional overlay feature for better comparison purpose Muse Oxidative Stress Reagent 1X Assay Buffer 1X Assay Buffer BA 11

Muse Ki67 Proliferation Kit (MCH100114). Ki67 is a prototypic cell cycle related nuclear protein, expressed by proliferating cells in all phases of the active cell cycle (G1, S, G2, and M phases), but is absent in the resting G0 phase. This characteristic makes Ki67 a good marker for the identification of proliferating cells. Uses assay reagent that is specific to Ki67 Simplified single color assay on Muse to detect proliferation index (% of cells proliferating in a population) Muse Ki67 Antibody Muse Isotype Control 5X Assay Buffer 5X Fixation Buffer Permeabilization buffer Optional histogram overlay of the isotype control (grey) 12

Autophagy 2 Assays 1) Muse Autophagy LC3-antibody based Kit (MCH200109) LC3 is a key protein indicated in autophagy which regulates traffic between autophagosome to lysosome. Provides the reagent for the disruption of the cell plasma membrane using a proprietary selective permeabilization solution. The selective permeabilization solution will extract cytosolic LC3 by flushing away during washing steps. LC3 translocated into the autophagosome is protected from the extraction and remains intact inside autophagosome, thereby allowing its fluorescence to be measured by flow cytometry. Anti-LC3 mouse monoclonal antibody conjugated to Alexa Fluor 555 is used to measure and track the levels of LC3 within the cell 2) Muse Autophagy Reporter Cell Line 13 (MCH200110) mrfp-lc3 reporter cell line for comparative studies constitutively produces LC3 Autophagy LC3-antibody based Kit 1) Muse LC3 Antibody Muse Reagent Detection Pack 5X Assay Buffer 2) mrfp-lc3 Reporter Cell Line plus Autophagy Reagent Pack

Cell Signalling Assays P P P

Muse H2A.X Activation Dual Detection Kit (MCH200101) This two color kit is designed to detect the extent of Histone H2A.X pathway activation by measuring H2A.X phosphorylation relative to the total H2A.X expression in any given cell population. Includes two directly conjugated antibodies, a phosphospecific anti-phospho-histone H2A.X (Ser139)-Alexa Fluor 555 and an anti-histone H2A.XPECy5 conjugated antibody to measure total levels of Histone H2A.X. The levels of both the total and phosphorylated protein can be measured simultaneously in the same cell, resulting in a normalized and accurate measurement of H2A.X activation after stimulation. A dot plot displaying cells which are inactivated, non-expressing, and activated. A bar graph illustration of the same data, calculating the expression of each cell population as a percentage (%). Anti-phospho-Histone H2A.X (Ser139), Alexa Fluor 555; Anti-H2A.X, PECy5 5 X Assay Buffer 1 X Fixation Buffer 1 X Permeabilization Buffer 15

Muse MAPK Activation Dual Detection Kit (MCH200104) This two color kit is designed to measure the extent of MAPK phosphorylation relative to the total MAPK expression in any given cell population. Two directly conjugated antibodies, a phosphospecific anti-phospho-erk1/2 Thr202/Tyr204, Thr185/Tyr187)-Phycoerythrin and an anti-erk1/2- PECy5 conjugated antibody to measure total levels of ERK. The levels of both the total and phosphorylated protein can be measured simultaneously in the same cell, resulting in a normalized and accurate measurement of MAPK activation after stimulation. Moreover, simultaneous measurement of both total and phospho-erk1/2 confirms target specificity of the phosphorylation event. A dot plot displaying cells which are inactivated, non-expressing, and activated. A bar graph illustration of the same data, calculating the expression of each cell population as a percentage (%). Anti-phospho-ERK1/2 (Thr202/Tyr204, Thr185/Tyr187), PE; Anti-ERK1/2, PECy5 5 X Assay Buffer 1 X Fixation Buffer 1 X Permeabilization Buffer 16

Muse EGFR-RTK Activation Dual Detection Kit (MCH200102) This two color kit is designed to detect the extent of EGFR pathway activation by measuring EGFR phosphorylation relative to the total EGFR expression in any given cell population. Two directly conjugated antibodies, a phosphospecific anti-phospho-egfr (Tyr1173)-Alexa Fluor 555 and an anti-egfr-pecy5 conjugated antibody to measure total levels of EGFR expression. The levels of both the total and phosphorylated protein can be measured simultaneously in the same cell, resulting in a normalized and accurate measurement of EGFR activation after stimulation. Moreover, simultaneous measurement of both total and phospho-egfr confirms target specificity of the phosphorylation event. A dot plot displaying cells which are inactivated, non-expressing, and activated. A bar graph illustration of the same data, calculating the expression of each cell population as a percentage (%). 17 Anti-phospho-EGFR (Tyr1173), Alexa Fluor 555; Anti-EGFR, PECy5 5 X Assay Buffer 1 X Fixation Buffer 1 X Permeabilization Buffer

Muse PI3K Activation Dual Detection Kit (MCH200103) This two color kit is designed to detect the extent of Akt phosphorylation relative to the total Akt expression in any given cell population. Two directly conjugated antibodies, a phosphospecific anti-phospho-akt (Ser473), Alexa Fluor 555 and an anti-akt, PECy5 conjugated antibody to measure total levels of Akt. The levels of both the total and phosphorylated protein can be measured simultaneously in the same cell, resulting in a normalized and accurate measurement of PI3K activation after stimulation. Moreover, simultaneous measurement of both total and phospho-akt confirms target specificity of the phosphorylation event. A dot plot displaying cells which are inactivated, non-expressing, and activated. A bar graph illustration of the same data, calculating the expression of each cell population as a percentage (%). Anti-phospho-Akt (Ser473), Alexa Fluor 555 Anti-Akt/PKB, PECy5 5 X Assay Buffer 1 X Fixation Buffer 1 X Permeabilization Buffer 18

Muse Bcl-2 Activation Dual Detection Kit (MCH200105) This two color kit is designed to detect the extent of Bcl-2 pathway activation by measuring Bcl-2 phosphorylation relative to the total Bcl-2 expression in any given cell population Two directly conjugated antibodies, a phosphospecific anti-phospho-bcl-2 (Ser70)-Alexa Fluor 555 and an anti-bcl-2-pecy5 conjugated antibody to measure total levels of Bcl-2 expression. The levels of both the total and phosphorylated protein can be measured simultaneously in the same cell, resulting in a normalized and accurate measurement of Bcl-2 activation after treatment. Moreover, simultaneous measurement of both total and phospho-bcl-2 confirms target specificity of the phosphorylation event. A dot plot displaying cells which are inactivated, non-expressing, and activated. A bar graph illustration of the same data, calculating the expression of each cell population as a percentage (%). Anti-phospho-Bcl-2 (Ser70)-Alexa Fluor 555 Anti-Bcl-2-PECy5 5 X Assay Buffer 1 X Fixation Buffer 1 X Permeabilization Buffer 19

Muse Multi-Color DNA Damage Kit (MCH200107) The kit measures the levels of two key signaling nodes along the DNA damage signaling pathway, H2A.X and Ataxiatelangiectasia mutated kinase (ATM). ATM is the main kinase activated in response to doublestranded DNA breaks. Once activated, ATM phosphorylates a number of downstream factors, including Histone H2A.X. Phosphorylation of H2A.X, at serine 139 by ATM is an important indicator of DNA damage. Two directly conjugated antibodies, a phosphospecific ATM (Ser1981)-PE and a phospho-specific Histone H2A.X-PECy5 conjugated antibody measure the extent of DNA damage in testing samples. This two color kit is designed to detect the phosphorylation state of ATM and Histone H2A.X simultaneously by flow analysis. 20 Anti-phospho-ATM (Ser1981), PE Anti-phospho-Histone H2A.X (Ser139), PECy5 5 X Assay Buffer 1 X Fixation Buffer 1 X Permeabilization Buffer

Muse PI3K/MAPK Dual Pathway Activation Kit (MCH200108) Evaluation of both the PI3K and MAPK signaling pathways simultaneously. There is evidence that these two key signaling pathways cross-talk downstream along the signaling cascade, which can potentially influence the activation of one another. Two directly conjugated antibodies, a phospho-specific Akt (Ser473)-Alexa Fluor 555 and a phospho-specific ERK1/2 (Thr202/Tyr204, Thr185/Tyr187)-PECy5 conjugated antibody assess the activation of both the PI3K and MAPK signaling pathways simultaneously in testing samples. 21 Anti-phospho-Akt (Ser473), Alexa Fluor 555 Anti-phospho-ERK1/2 (Thr202/Tyr204,Thr185/Tyr187), PECy5 5 X Assay Buffer 1 X Fixation Buffer 1 X Permeabilization Buffer

Immunology Assays CD8 T cells Mono CD4 T cells B cells Tregs NK Cells

Muse Human CD4 T Cell Kit (MIM100101) Detection and identification of lymphocytes and CD4 T lymphocytes in either whole blood or PBMCs using a simplified no-wash assay. The CD4 antibody identifies human helper/inducer CD4+ T cell (HLA Class II reactive) and recognizes a 60,000 Da surface antigen. Monocytes also express CD4 but at a lower density, and have no co-expression of the other antibodies present in the anti-lymphocyte cocktail; hence can be distinguished from CD4 T cells in this kit. The assay uses an anti-lymphocyte cocktail that identifies the lymphocyte population, and a CD4 antibody that binds to the CD4 cells. Non-CD4 Lymphocytes Negative CD4 T Lymphocytes CD4 Non- Lymphocytes Muse Human CD4 T Cell Cocktail 1 X Assay Buffer BA 1 X Lysing Solution 23

Muse Human CD8 T Cell Kit (MIM100102) Detection and identification of lymphocytes and CD8 T lymphocytes in either whole blood or PBMCs using a simplified no-wash assay. The CD8 antibody allows the identification of CD8, a 68- kda transmembrane glycoprotein expressed by class I major histocompatibility complex restricted, mature suppressor/cytotoxic T cells, the great majority of cortical thymocytes and approximately 30% of medullary thymocytes. In addition a proportion of γδ T cells and NK cells express CD8. Inclusion of several proprietary antibodies in the anti-lymphocyte cocktail allows for the unique identification of the CD8 cytotoxic T cells. The assay uses an anti-lymphocyte cocktail that identifies the lymphocyte population, and a CD8 antibody that binds to the CD8 cells. CD8 T Lymphocytes PROFILE CD8 (-) T & B Lymphocytes Negative CD8 CD8(+) T Lymphocytes CD8 (+) Cells Muse Human CD8 T Cell Cocktail 1 X Assay Buffer BA 1 X Lysing Solution 24

Muse Human B Cell Kit (MIM100103) Detection and identification of lymphocytes and CD19 B lymphocytes in either whole blood or PBMCs using a simplified no-wash assay. CD19 is considered to be a characteristic B cell marker and therefore commonly used in the routine immunophenotyping of B cells. Monoclonal antibodies clustered as CD19 detect all peripheral blood B cells. The assay uses an anti-lymphocyte cocktail that identifies the lymphocyte population, and a CD19 antibody that binds to the B cell population. Non-B Lymphocytes (CD19-) Negative B Lymphocytes (CD19+) Non-Lymphocytes (CD19+) Muse Human B Cell Cocktail 1 X Assay Buffer BA 1 X Lysing Solution 25

Muse Human Lymphocyte CD69 Kit (MIM100104) Detection and identification of lymphocytes and CD69 lymphocytes in either whole blood or PBMCs using a simplified no-wash assay. CD69 is a disulfide-linked dimeric structure of two phosphorylated polypeptide chains (27 kda and 33 kda) that are differentially glycosylated forms of the same core protein. CD69 (also known as activation inducer molecule [AIM]), is the earliest inducible surface antigen expressed on lymphocytes after T- or B-cell activation and is absent from resting lymphocytes. The assay uses an anti-lymphocyte cocktail that identifies the total lymphocyte population, and a CD69 antibody that identifies activation marker CD69. The kit also includes an isotype control antibody. negative CD69+ Lymphocytes 26 Muse Human Lymphocyte Cocktail Human CD69-PE Antibody Isotype Control Mouse IgG1-PE 1 X Assay Buffer BA 1 X Lysing Solution

Muse Human Lymphocyte CD25 Kit (MIM100105) Detection and identification of lymphocytes and CD25 lymphocytes in either whole blood or PBMCs using a simplified no-wash assay. CD25 (IL-2 receptor α-chain) is an mid to late activation marker expressed on the surface of activated lymphocytes including T and B lymphocytes and NK cells. It is also expressed constitutively on the surface of small subpopulations of T cells. The assay uses an anti-lymphocyte cocktail that identifies the total lymphocyte population, and a CD25 antibody that identifies activation marker CD25. The kit also includes an isotype control antibody. negative CD69+ Lymphocytes 27 Muse Human Lymphocyte Cocktail Human CD25-PE Antibody Isotype Control Mouse IgG1-PE 1 X Assay Buffer BA 1 X Lysing Solution

Nucleic Acid Assays

SmartFlare RNA Detection Probes Detection on Muse Cell Analyzer SmartFlare RNA detection reagent is a novel probe capable of detecting specific mrnas and mirnas in live cells. This technology allows for carrier-free cellular endocytosis of the reagent, followed by detection and relative quantitative analysis of RNA levels in individual cells Each SmartFlare probe consists of a gold nanoparticle conjugated to multiple copies of a doublestranded oligonucleotide, in which one strand (the reporter strand ) bears a fluorophore that is quenched by its proximity to the gold core. When the nanoparticle comes into contact with its target RNA, the target RNA binds to its complementary capture strand and displaces the reporter strand. The reporter strand, whose Cy3 fluorophore is now unquenched, fluoresces and can be detected using the Muse Cell Analyzer. SmartFlare RNA detection probes conjugated with Cyanine (Cy3) fluorescent dye are optimized on the Muse Cell Analyzer. Data generated using the Muse provides the RNA expression levels of target genes and the percentage of expressing cells. Unflared - cells are incubated in media alone Scrambled sequence unspecific SmartFlare Target - SmartFlare Cy3 probes 29