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Flow Cytometry Products and Capabilities Guides | Thermo Fisher Scientific - US
Flow cytometry capabilities guide Sample preparation | Fluorophore selection | Flow cytometry antibodies and assays | Attune NxT Flow Cytometer | PrimeFlow RNA Assay | Fluorophore and reagent poster Getting started Flow cytometry enables simultaneous analysis of multiple proteins, gene expression, and cell functions such as oxidation, viability, cell cycle, apoptosis, and proliferation from an individual cell. This technology makes it possible to obtain a statistically relevant amount of data by combining information from individual cells in order to gain insight into a heterogeneous sample. Whether you are identifying cell subpopulations or investigating cell functions, flow cytometry can make significant contributions to moving your research forward. Building a flow cytometry experiment often requires combining products into a multicolor panel. Use this guide to understand the basics of InvitrogenTM eBioscienceTM flow cytometry antibodies and InvitrogenTM flow cytometry assays and reagents. Then see how example panels are run on flow cytometers, including the InvitrogenTM AttuneTM NxT Flow Cytometer, in the following areas: · Immunology · Neuroinflammation · Inflammation · Gene editing · Immuno-oncology · Microbiology · Solid-tumor cancers Flow cytometry workflow--what you will need Sample preperation Immunophenotyping with antibodies Buffer selection Cell function assays Experimental controls Running experiment Live cell Dead cell Figure 1. Flow cytometry workflow. Planning your workflow in advance as outlined will help generate a successful experiment. Find out more about multipurpose flow cytometry experiments at thermofisher.com/flowcytometry 2 Sample preparation: reagents for immune cell activation Stimulation or treatment of cells is usually required for activation of immune cells to proliferate and differentiate into mature cell types (Figure 2). Activated cells often express higher levels of transcription factors, cytokines, chemokines, and other mediators detected by flow cytometry. Choosing the appropriate activating reagent will depend on (1) cell type, (2) expression and kinetics of the protein of interest, and (3) experimental conditions. We offer an expansive list of high-quality cell stimulation products that include: · Functional-grade antibodies and recombinant proteins to stimulate many types of immune cells · Reagents in appropriate preservative-free buffers with extremely low endotoxin levels to use in cell culture · The InvitrogenTM eBioscienceTM Cell Stimulation Cocktail at a ready-to-use concentration A CD28 receptor B Ionomycin C Con A D LPS E M-CSF CD14 CSF-1R CD3 receptor CD28/CD3 TCR-mediated activation and co-stimulation PMA Ionomycin and PMA Leucocyte activation and stimulation Con A Mitogen-mediated activation of leukocytes LPS Leukocytes activation M-CSF Macrophage monocyte di erentiation and survival Figure 2. Cell stimulation reagents. (A) Functional-grade antibodies (e.g., anti-CD3 and anti-CD28) or InvitrogenTM DynabeadsTM magnetic beads for T cell activation and expansion. (B) InvitrogenTM eBioscienceTM Cell Stimulation Cocktail comprising phorbol 12-myristate 13-acetate (PMA), a protein kinase activator, and ionomycin, a calcium ionophore, stimulate T cells to produce interferon-gamma (IFN-), tumor necrosis factor-alpha (TNF-), interleukin-2 (IL-2), and interleukin-4 (IL-4). (C) Concanavalin A (Con A) induces T cell activation and proliferation. (D) Monocytes can be activated by lipopolysaccharide (LPS) to secrete interleukin-6 (IL-6), interleukin-10 (IL-10), or TNF-. (E) Macrophage colony-stimulating factor (M-CSF) is a growth factor that regulates the proliferation, differentiation, and functional activation of monocytes' differentiation into macrophages. Example: T cell activation T cells require external signals for differentiation and expansion from a quiescent state (Figure 3). PMA and ionomycin or anti-CD3 and anti-CD28 antibodies are recommended to upregulate intracellular transcription factors for detection. Time-course profiling of cells with the cell-stimulating reagents is recommended, since cytokines have different kinetics and/or expression levels. IFN---APC IFN---APC Identification of human Th17 cells within a CD4+ T cell population Unstimulated Stimulated Find out more at thermofisher.com/flow-assays FITC CD4FITC Figure 3. Identification of human Th17 cells within a CD4+ T cell population. Normal human peripheral blood cells were unstimulated (left) or stimulated with eBioscience Cell Stimulation Cocktail plus protein transport inhibitors (500X) (right). Cells were fixed and stained intracellularly with InvitrogenTM antihuman CD4 APC and antihuman IFN- conjugated to eBioscienceTM PE-eFluorTM 610 dye, using the InvitrogenTM eBioscienceTM Intracellular Fixation and Permeabilization Buffer Set and protocol. Cells in the lymphocyte gate were used for analysis. 3 Immunophenotyping with flow cytometry antibodies A multicolor flow cytometry panel uses two or more primary conjugated antibodies to identify single cells by detecting multiple antigens. The goal of the panel is to get the maximum signal for effective visualization of cell populations. Use this section of the guide to aid in the selection of antibodies. Flow cytometry antibodies cover: · CD markers · Transcription factors · Cytokines, chemokines, and growth factors · Signaling pathway markers, including phosphoproteins Marker selection Select from one of the largest portfolios of primary conjugated antibodies specifically developed for flow cytometry applications. Each flow cytometry antibody search result contains data plots gathered from internal antibody validation* testing and published customer data accessible online. Use this online search tool to determine which antibody is applicable to find your cell population (Figure 4). Step 1: Enter antibody needs Step 2: Choose an InvitrogenTM eBioscienceTM antibody for your applications Our flow cytometry antibodies are conjugated to different fluorophores to allow for use on any instrument. These fluorophores simplify the optimization of panel design because of flexible dye selection for reduced spectral overlap. Choose dyes based on: · Laser and filter configuration of the flow cytometer · Expression level or abundance of the target protein · Fluorophore brightness · Fluorescence excitation emission spectra Example: selecting the right fluorophore Fluorophore selection is important for finding your cell of interest. Pick fluorophores with less spectral overlap to clearly identify two populations (Figure 5). Match brighter fluorophores with less abundant targets, and dimmer fluorophores with abundant targets for greater signal separation. * The use or any variation of the word "validation" refers only to research use antibodies that were subject to functional testing to confirm that the antibody can be used with the research techniques indicated. It does not ensure that the product(s) was validated for clinical or diagnostic uses. Figure 4. Antibody search tool to find information and purchase antibodies. (Left) Antibody application data from customer publications and internal testing data. (Right) A list of antibodies can be purchased, or saved and shared for later use. Relative intensity (%) CD56 APC Relative intensity (%) CD56 APC CD3 APC-eFluor 780 CD56 APC 100 75 50 25 0 300 400 500 600 700 Wavelength (nm) 800 900 105 10 10³ 0 0 103 104 105 CD3 APC-eFluor 780 CD3 APC-eFluor 450 CD56 APC 100 75 50 25 0 300 400 500 600 700 Wavelength (nm) 800 900 105 NKT cells 10 10³ 0 0 103 104 105 CD3 APC-eFluor 450 Figure 5. Normal human peripheral blood cells were stained with antihuman CD3 antibody conjugated with InvitrogenTM eBioscienceTM APCeFluorTM 780 dye (left) or eFluorTM 450 dye (right), as well as antihuman CD56 antibody conjugated with APC dye. Cells in the lymphocyte gate were used for analysis. Find your flow cytometry antibodies at thermofisher.com/flowantibodies 4 Table 1. Comprehensive list of available fluorophores based on their usage, benefits, and intended applications. Family Type Original Pacific dyes Organic dyes--small, stable molecules Alexa Fluor dyes eFluor organic dye Original Large, protein-based molecules Tandem dyes Polymer dyes--recent dye Super Bright dyes and innovation their tandems Benefit InvitrogenTM fluorophore · FITC is cost-efficient FITC · Some of the dimmest dyes Pacific Blue Pacific Orange Alexa Fluor 405 Alexa Fluor 488 · Photostable dyes that range across the visible spectrum Alexa Fluor 532 · Used in flow cytometry and imaging Alexa Fluor 561 · Named for their excitation wavelengths Alexa Fluor 647 Alexa Fluor 660 Alexa Fluor 700 · Engineered for detection for flow cytometry · Named for their emission wavelength eFluor 450 eFluor 506 eFluor 660 · Cost-efficient · Some of the brightest dyes available APC PE PerCP APC-Cyanine5 APC-Cyanine7 PE-Cyanine5 (TRI-COLOR) PE-Cyanine5.5 PE-Cyanine7 · Dyes occupy different channels from the donor molecule, and this can be used to build larger panels PETexas Red PerCP-Cyanine5.5 PEAlexa Fluor 610 PEAlexa Fluor 700 APCAlexa Fluor 750 PEeFluor 610 PerCPeFluor 710 APCeFluor 780 · Excited by the 405 nm violet laser Super Bright 436 · Minimal spillover into other channels Super Bright 600 · Add Super Bright Complete Staining Buffer (Cat. No. Super Bright 645 SB-4401-42) when using two or more polymer dyes to Super Bright 702 lower background levels Super Bright 780 Creating a flow cytometry panel The InvitrogenTM Flow Cytometry Panel Builder is a free online tool to help select antibody conjugates and reagents for a multicolor flow cytometry panel (Figure 6). This allows for improved panel design with greater separation and detection of individual cell populations of interest. With this tool, you can: · Create a new immunophenotyping experiment or add antibodies and reagents to an existing panel · Check fluorophore emission spectra with the built-in SpectraViewer · Export an ExcelTM document with your antibody choices, or order directly Step 1: Cytometer Step 2: Antigens Step 3: Panel Builder Step 4: Products Step 5: Summary Figure 6. The Flow Cytometry Panel Builder simplifies experimental design with a 5-step strategy. Plan your experiment at thermofisher.com/flowpanel 5 Application spotlight--immunophenotyping on a spectral flow cytometer · Standard panel design rules apply · InvitrogenTM fluorescent probes and reagents are suitable for all cytometry instrumentation, including spectral flow cytometers; use fluorophores designed for use with spectral instruments including Alexa Fluor 561 and Alexa Fluor 660 dyes · Many previously incompatible labeling dyes and functional reagents, including PerCP and PerCP eFluor 710 dyes, can now be used together in your expanded multicolor application · Expand your panel with Alexa Fluor 532, Pacific Orange, eFluor 450, and Super Bright 436 labels Target selection CD3 Fluorophore selection Add viability dye or cell functional assay Intensity Look for unique signatures and minimal overlap PerCP 106 105 104 103 0 V2 V4 V6 V8 V10 V12 V14 V16 B2 B4 B6 B8 B10B12 B14 R2 R4 R6 R8 Channel Violet detectors Blue detectors Red detectors 400 nm850 nm 500 nm900 nm 650 nm900 nm Conjugated antibodies Live Dead Intensity PerCP-eFluor 710 106 105 104 103 0 V2 V4 V6 V8 V10 V12 V14 V16 B2 B4 B6 B8 B10B12 B14 R2 R4 R6 R8 Channel Violet detectors 400 nm850 nm Blue detectors Red detectors 500 nm900 nm 650 nm900 nm Violet laser (405 nm) Blue laser (488 nm) Red laser (635 nm) Super Bright 436 eFluor 450 eFluor 506 Pacific Orange Super Bright 600 Super Bright 645 Super Bright 702 Brilliant Violet 785 FITC Alexa Fluor 532 P E PE-eFluor 610 PE-Cy5 PE-Cy5.5 PerCP-eFluor 710 PE-Cy7 APC Alexa Fluor 647 Alexa Fluor 700 Alexa Fluor 780 Super Bright 436 eFluor 450 eFluor 506 Pacific Orange Super Bright 600 Super Bright 645 Super Bright 702 Brilliant Violet 785 FITC Alexa Fluor 532 PE PE-eFluor 610 PE-Cy5 PE-Cy5.5 PerCP-eFluor 710 PE-Cy7 APC Alexa Fluor 647 Alexa Fluor 700 APC-eFluor 780 * All spectral flow cytometry data shown were generated by Cytek Biosciences on a CytekTM AuroraTM spectral flow cytometer three-laser system and analyzed using SpectroFloTM software. Table 2. Staining spread matrix of 20 Invitrogen fluorophores that can be used simultaneously on a three-laser spectral flow cytometer.* All fluorophores were compared using anti-CD4 antibody conjugates to demonstrate the level of spread among dyes. The fluorophore in each row impacts the resolution of the fluorophore in each column. Although all dyes in the matrix can be used together, the darker red shading means one fluorophore has increased spread into the other and needs closer attention during panel design and data interpretation. Find out more about panel design at thermofisher.com/spectralflowcytometry 6 Buffer selection: fixation and permeabilization reagents Fixatives are necessary for saving samples to be used later or for looking at intracellular or intranuclear targets. Ready-to-use fixation kits are optimized for flow cytometry applications. Benefits of using these kits include the following: · Methods used to stain cells take into consideration the location of the target proteins · The fixation and permeabilization procedure keeps the morphological lightscattering characteristics of the cells intact · The reagents in the kits help reduce background staining Table 3. Cell staining workflow. Cell-surface staining (CD markers) Stain surface proteins Fix cells Permeabilize cells Stain cytoplasmic proteins Stain nuclear proteins * Cytoplasmic proteins may be stained with a nuclear staining kit, but it may not be optimal. Cytoplasmic staining (cytokines) Nuclear and cytoplasmic staining (cytokines and transcription factors) * Table 4. Flow cytometry buffer and reagent selection guide. Staining buffer Description Location eBioscience Flow Cytometry Staining Buffer Cell-surface markers are often used to identify cell types. Permeabilization techniques can damage or denature cell-surface antigens and prevent antibodies from binding to surface epitopes. It is advisable to stain for cell-surface antibodies separately. Cell-surface markers can also be stained first, and then protocols for cytoplasmic or nuclear staining should be followed. Cell surface InvitrogenTM FIX & PERMTM Cell Permeabilization Kit (RUO and clinical researchgrade) or Intracellular Fixation and Permeabilization Buffer Set (RUO) Cytoplasmic proteins can include cytokines, organelles, and cytoplasmic transcription factors. These proteins are easily accessible with gentle fixation and light permeabilization. Fixation of cytoplasmic proteins often requires a crosslinking agent to have the protein trapped within the cell. Cytoplasm eBioscience Foxp3/ Transcription Buffer Set Transcription factors, DNA-binding proteins, and modified proteins make up the bulk of nuclear proteins. A quick fixation combined with a stringent permeabilization allows antibodies to penetrate into the nucleus. Fixation reagents can include either crosslinking agents or organic solvents. This type of protocol is also appropriate when examining proteins found both in the cytoplasm* and nucleus. Nucleus * Cytoplasmic proteins may be stained with a nuclear staining kit, but it may not be optimal. Find out more about buffers at thermofisher.com/flow-sample 7 Cell function assays: dyes and reagents Flow cytometry is more than just panels with antibodies. Fluorophore reagents can be used to label cell functionalities such as viability and mitochondrial oxidation. These reagents and assays can be incorporated into a flow cytometry panel just like a flow cytometry antibody. Use the chart below to determine which assays can be incorporated into a panel (Figure 7). Cell function workflow with flow cytometry reagents What type of applications are you using in flow cytometry? Cell proliferation Population doublings CellTrace Reagents Cell cycle Live cell Vybrant DyeCycle stains Apoptosis Annexin conjugates, CellEvent Caspase3/7 Green DNA replication Click-iT Plus EdU, Click-iT EdU, BrdU Fixed cell FxCycle stains, CellEvent Senescence, Propidium lodide, 7-Aminoactinomycin D, (7-AAD) SYTOX dyes Figure 7. Cell function workflow with flow cytometry reagents. Viability Fixable LIVE/DEAD stains, eFluor fixable viability dyes Not fixed SYTOX stains, 7-AAD stain, DRAQ7 dye, Propidium lodide Metabolism Mitochondrial probes MitoProbe TMRM, JC-1, MitoProbe DiOC2(3), MitoProbe DilC1(5), MitoProbe Transition Pore Oxidative stress Total ROS Assay, CellROX stains, Hypoxia Green reagent Calcium indicators eBioscience Calcium Sensor Dye eFluor 514, Indo-1 AM Cell viability Cell viability assays can be used to simply distinguish between live and dead cell populations, to correlate with other cell functions or treatments, or to exclude dead cell populations from analyses. Our assays are all one- or two-step processes and can be used in cell sorting or analysis applications. Membrane dyes to characterize extracellular vesicles (EVs) Uniformly label a population of EVs from cell culture. These reagents stain lipids, which is useful for EV detection. · Lipophilic styryl dye: InvitrogenTM FMTM Dye · Long-chain lipophilic carbocyanine dyes: InvitrogenTM DiI, VybrantTM CM-DiI (fixable), DiO, and DiD dyes, or VybrantTM Multicolor Cell Labeling Kit · InvitrogenTM Di-8-ANNEPS dyes Table 5. Cell viability dyes selection guide. Laser UV 405 nm 488 nm 532 nm 561 nm 633/5 nm Live cell/nonfixable stains Fixable stains SYTOX Blue (450/50*) LIVE/DEAD fixable blue dead cell stain (350/40*) LIVE/DEAD fixable violet dead cell stain (450/40*) SYTOX Blue (450/50*) LIVE/DEAD fixable aqua dead cell stain (530/50*) LIVE/DEAD fixable yellow dead cell stain (585/42*) SYTOX Green (530/30*) LIVE/DEAD fixable green dead cell stain (530/30*) SYTOX AADvanced (>650*) LIVE/DEAD fixable red dead Propidium Iodide (~617*) cell stain (>650 or 600/20*) SYTOX Orange (585/42*) LIVE/DEAD fixable red SYTOX AADvanced (>650*) dead cell stain (>650 or red Propidium Iodide (~617*) bandpass*) LDS 751 (700/20*) LIVE/DEAD fixable red dead cell stain (>650 or red*) SYTOX Red (660/20*) LIVE/DEAD fixable far-red dead cell stain (660/20*) LIVE/DEAD fixable near-IR dead cell stain (780/60*) * Recommended filters (nm). Find out more at thermofisher.com/flow-assays 8 Example: avoid inaccurate analysis with LIVE/DEAD assay When choosing a viability dye to stain cells post-fixation, it is important to select one that is retained in the cell post-fixation and preserves the staining pattern. Exclusion of the dead cells from the data allows cleaner A Lymphocyte gate B Viability gate separation and identification of cell populations (Figure 8). InvitrogenTM LIVE/DEADTM Fixable Dead Cell Stains are fixable viability dyes that help to ensure accurate assessment of cell viability in samples after fixation and/or permeabilization (Figure 9). C D FSC-A FSC-A CD4-Cyanine5.5-PE fluorescence CD4-Cyanine5.5-PE fluorescence SSC LIVE/DEAD Fixable Violet fluorescence CD8-Q705 fluorescence CD8-Q705 fluorescence Figure 8. Exclusion of dead cells eliminates staining artifacts from analysis. After the application of a lymphocyte gate (A), live and dead cells were discriminated (B) using the InvitrogenTM LIVE/DEADTM Fixable Violet Dead Cell Stain Kit (Cat. No. L34963). Note the significant number of dead cells despite a scatter gate. Subsequent analysis of dead cells (C) and live cells (D) shows the dramatic difference in apparent phenotypes between the two cell populations. Reprinted from Perfetto SP, Chattopadhyay PK, and Lamoreaux L et al. (2006) J Immunol Methods 313:199208, with permission from Elsevier. Application spotlight--bacterial cell viability workflow Flow cytometry methods can shorten bacterial phenotyping and counting time. · A serial dilution is not necessary--just take stained sample, dilute, and analyze · To obtain a single bacterial cell suspension, beverages and solid foods should be weighed and homogenized · InvitrogenTM LIVE/DEADTM BacLightTM kits can be used to quickly determine bacterial cell viability Bacterial source Stock culture Beverage Food Depending on source, subculture or filter the cell suspension Stain sample with LIVE/DEAD BacLight Bacterial Viability Kit Propidium iodide Dead SYTO 9 Live Injured Run on a flow cytometer SYTO Fluorescence Pasteurella multocida 105 104 103 102 102 103 104 105 Propidium Iodide Fluorescence Figure 9. Pasteurella multocida bacteria labeled with LIVE/DEAD BacLight kit stains for 15 min. Sample was analyzed on the Attune NxT Flow Cytometer. Find out more about cell viability dyes at thermofisher.com/flow-cellviability 9 Cell proliferation Cell proliferation analysis is important for drug development and cell tracing applications. Proliferation measurements are typically made based on average DNA content or on cellular metabolism parameters. Assays can report either total live cell numbers or measure DNA synthesis in single cells. We offer dyes, kits, and antibodies to track proliferation. Use our guide to find suitable reagents for flow cytometry assays or multicolor panels. Table 6. Flow cytometry reagent selection guide for cell proliferation assays. Product Target Fixable Live-cell analysis Click-iT Plus EdU Flow Cytometry Incorporation into newly Assay Kits synthesized DNA Yes Yes BrdU Incorporation into newly synthesized DNA Yes Yes CellTrace Cell Proliferation Kits Lysine-containing proteins Yes Yes Ki-67 antibody Nuclear protein expressed in proliferating cells Yes Yes Minichromosome maintenance (MCM2) antibody Nuclear protein expressed in proliferating cells Yes No Proliferating cell nuclear antigen (PCNA) antibody Nuclear protein expressed in proliferating cells Yes No Application Cell proliferation Cell proliferation Generational analysis Cell proliferation and cell cycle Cell proliferation and cell cycle Cell proliferation and cell cycle Example: generational tracing with CellTrace reagent InvitrogenTM CellTraceTM reagents track cell division by analyzing cell subsets for dye dilution in successive generations (Figure 10). When cells proliferate, the fluorescence of each proliferating generation is half as bright compared with the previous generation. The CellTrace reagents help to monitor and visualize distinct generations of proliferating cells. With these reagents, you can observe one uniformly labeled cell population for each generation. Number of cells counted 54 "CellTrace Violet is the best reagent for tracking proliferation in any amenable cell type by 41 fluorescent dye dilution and flow cytometry. Compared to CFSE, which is cytotoxic to cells 27 when used at higher concentrations, CellTrace Violet labels cells brightly, with low toxicity and is faithfully distributed to daughter cells, ensuring the 14 best possible peak resolution." 0 10 10¹ 10² 10³ 10 10 CellTrace Violet Fluorescence Andrew Filby, Flow Cytometry Core Facility Manager and ISAC SRL Emerging Leader, Newcastle University Figure 10. Tracing cell divisions with CellTrace reagent. Human peripheral blood lymphocytes were harvested and stained using the InvitrogenTM CellTraceTM Violet Cell Proliferation Kit. The violet peaks represent successive generations of cells stimulated with InvitrogenTM mouse antihuman CD3 and interleukin-2, and grown in culture for 7 days. The peak outlined in black represents cells that were grown in culture for 7 days with no stimulus. Find out more about cell proliferation reagents at thermofisher.com/flow-cellproliferation 10 RNA detection by flow cytometry With the novel InvitrogenTM PrimeFlowTM RNA Assay, scientists can now reveal the dynamics of RNA and protein expression simultaneously within millions of single cells (Figure 11). This assay employs a proprietary fluorescence in situ hybridization (FISH) and branched DNA (bDNA) amplification (Figure 12) technique for simultaneous detection of up to four RNA transcripts labeled with InvitrogenTM Alexa FluorTM 488, Alexa FluorTM 568, Alexa FluorTM 647, and Alexa FluorTM 750 dyes, in a single cell using a standard flow cytometer. RNA detection may be combined with intracellular and cell-surface antibody staining to elevate the understanding of single-cell dynamics to a new dimension. Novel product applications: · Unmask gene expression heterogeneity at the single-cell level · Correlate RNA and protein levels in the same cell · Detect noncoding RNA, microRNA (miRNA), and long noncoding RNA (lncRNA) · Evaluate viral RNA in infected cells · Analyze mRNA expression when antibody selection is limited · Analyze up to four RNA transcripts simultaneously · Detect telomere DNA miR-mi1R4-61a46AlaeAxlaexFlauFloruo6r46747 ArAgr1g1mmRRNNAAAlAleexxaaFlFluuoorr 448888 CxCclx1cl313mRmNRANAAlAelxeaxaFlFluuoror775500 RetRne1tanlamRmNRANAAlAelxeaxaFlFuluoror556688 FF44/8/800eeFFluluoorr 445500 FF44//8800 eeFluor 45500 F4//8800 eeFFluluoorr445500 F4/80 eFFlluuoorr445500 Figure 11. PrimeFlow RNA Assay detection of miR-146a, Arg1 mRNA, Cxcl13 mRNA, and Retn1a mRNA in mouse peritoneal cells. C57BI/6 mouse resident peritoneal exudate cells were analyzed using the PrimeFlow RNA Assay. Cells were stained with InvitrogenTM eBioscienceTM AntiMouse F4/80 eFluor 450 and AntiMouse CD11b PE-Cyanine7 antibodies, then fixed and permeabilized using PrimeFlow RNA Assay buffers and protocols. Cells were then hybridized to label RNA with InvitrogenTM Type 1 Human/Mouse miR146a Alexa Fluor 647, Type 4 Mouse Arg1 Alexa Fluor 488, Type 6 Mouse Cxcl13 Alexa Fluor 750, and Type 10 Mouse Retn1a Alexa Fluor 568 target probes. Viable CD11b+ cells were used for analysis. Data show that both small peritoneal macrophages (SPM, F4/80) and large peritoneal macrophages (LPM, F4/80+) were positive for miR-146a. SPM expressed high levels of Retn1a (Relm-alpha) mRNA, whereas LPM were positive for Cxcl13 mRNA and expressed low levels of Arg1 mRNA. Sample preparation Label proteins with antibody (optional) Fix and permeabilize cells in suspension Label intracellular proteins with antibodies (optional) Gene 1 Gene 1 Gene 2 Gene G1 ene 2 Gene 1 Gene 2 Gene 2 Target hybridization Gene-specific label extenders (LE) Incubate cells with gene-specific probe sets Signal amplification Pre-amplifier Amplifier Hybridize with pre-amplifier and amplifier DNA Detection Fluorescent labeled probes Add labeled probes to cells Process cells using a flow cytometer CD8 mRNA Alexa Fluor 647 Suspension cells Gweinthe 1fixed RNA Gene 1 Gene 1 Gene 2 CD8 PE-Cyanine7 Figure 12. The PrimeFlow RGNenAe 2Assay workflow. The assay workflow contains several steps: antibody staining; fixation and permeabilization, including Gene 2 intracellular staining, if desired; and target hybridization with a target-specific probe set containing 2040 oligonucleotide pairs. Find out more about buffers at thermofisher.com/primeflow 11 Compensation and instrument beads Compensation beads for flow cytometry Emission profiles of fluorophores are broad, which can result in overlapping profiles that require compensation for signal correction. Compensation can be set using beads, particularly when cell samples are limited or when a positive population is needed. The latest generation of compensation beads Build flow cytometry panels with more accurate compensation using new InvitrogenTM UltraComp eBeadsTM Plus Compensation Beads. When a fluorophore-conjugated antibody is added to the beads, both positive and negative populations result. UltraComp eBeads Plus Compensation Beads now offer: · Increased species reactivity including rabbit- and human-origin antibodies (Figure 13) · Compatibility with fluorophores excited by ultraviolet (355 nm), violet (405 nm), blue (488 nm), green (532 nm), yellowgreen (561 nm), and red (633640 nm) lasers · Better compensation resolution for antibodies conjugated with InvitrogenTM eBioscienceTM Super Bright 780, Brilliant Violet 711, or Brilliant Violet 786 dyes Figure 13. Staining of UltraComp eBeads Plus Compensation Beads with 14 different antibody species. Beads were stained with 0.25 µg of each antibody and analyzed by flow cytometry. Table 7. InvitrogenTM antibody compensation beads. UltraComp UltraComp OneComp eBeadsTM Plus beads eBeadsTM beads eBeadsTM beads AbCTM Total Antibody ArCTM Amine Reactive Compensation Bead Kit* Compensation Bead Kit Application Immunophenotyping Cell viability assay Reactivity Human, rabbit, hamster, mouse, and rat antibodies Hamster, mouse, and rat antibodies with recognition of the kappa and lambda chains Hamster, mouse, rabbit, and rat antibodies LIVE/DEADTM fixable dead cell stains* Format One vial: dispense as a single drop 1 vial positive beads, 1 vial negative beads Laser compatibility Quantity Cat. No. Compatible with most standard lasers, UV to 633 nm; improved for polymer dye use from the violet laser 01-3333-41 01-3333-42 * Also applicable to similar amine-reactive dyes. Compatible with most standard lasers, UV to 633 nm 01-2222-41 01-2222-42 Compatible with most standard lasers, but not with UV or violet lasers Compatible with most standard lasers, UV to 633 nm 25 tests or 100 tests 01-1111-41 01-1111-42 A10513 A10497 A10628 A10346 GFP BrightComp eBeadsTM beads GFP expression; beads are present at 3 levels of GFP-like intensity GFP isoforms One vial: dispense as a single drop 488 nm 25 tests A10514 12 Counting beads Absolute cell counts is a method for quantifying cell concentration or absolute count of cells in a sample. Benefits of our absolute counting beads include: · Wide range of fluorophores to fit a broad spectrum (Figure 14) · Accommodates most cell sizes with increased percentage of singlets Count 400 CountBright Plus 200 CD19+ cells 0 10³ 0 10³ 10 10 10 CD19 APC-eFluor 780 Figure 14. CountBright Plus beads can be used with a broader range of fluorophores. CountBright Plus beads (red) can be detected simultaneously with cells stained with InvitrogenTM CD19 APC-eFluorTM 780 antibody (pink) in lysed whole blood when excited with an IR laser (808 nm) with an 840/20 nm emission filter. Table 8. InvitrogenTM absolute counting beads. CountBrightTM Plus beads* AccuCheckTM beads Parameters measured Cell concentration in sample · Cell concentration in sample · Pipetting accuracy Sample type Any type Whole blood Bead size 4 µm Bead A 6.40 µm Bead B 6.36 µm Range Ex: UV800 nm Em: 385860 nm Bead A Ex: 488 nm Em: 575585 nm Bead B Ex: 635 nm Em: 660680 nm Cat. No. C36995 PCB100 * The original InvitrogenTM CountBrightTM Absolute Counting Beads are still available, but not compatible with IR-excitable fluorophores. ** Stains all cells, so a pure bacterial sample is required for accurate results. LIVE/DEADTM BacLightTM Bacterial Viability and Counting Kit** · Viability · Bacterial concentration in sample Bacteria 6 µm Ex: 488 nm Em: 617 nm, 498 nm L34856 Calibration and size beads Instrument calibration is critical to collecting and analyzing accurate experimental data. Our beads are designed to help ensure robust flow cytometer performance. Table 9. InvitrogenTM calibration beads. Product Size calibration Instrument control Flow Cytometry Flow Cytometry Sub-micron Rainbow Calibration Size Calibration Kit Particle Size Reference Kit Particles Use Size reference Size reference Routine calibration of flow cytometers Emission No fluorescence Green fluorescence Bead size 6 sizes: 1.015 µm range 6 sizes: 0.022.0 µm Cat. No. F13838 F13839 400680 nm 3.03.4 µm A34305 Figure 15. ERF particles provide three fluorescence intensities. Alignment control Fluorescence standardization AlignflowTM Flow Cytometry AccuCheck ERF Alignment Beads Reference Particles Calibrate laser alignment Standardization and calibration for inter- and intra-instrument data comparisons 3 types: 400470 nm (for UV lasers), 515660 nm (for blue lasers), or 645680 nm (for red lasers) 415910 nm 2 sizes: 2.5 or 6.0 m diameter 3.2 M 2.5 m: A16502, A16500, A16501 6.0 m: A16505, A16503, A16504 A55950 Find out more about flow cytometry beads and controls at thermofisher.com/flow-controls 13 Sample analysis: Attune NxT Flow Cytometer, CytKick Autosamplers, and automation Run samples faster and achieve greater resolution--with little fear of sample loss due to clogging. The Attune NxT Flow Cytometer with InvitrogenTM CytKickTM or CytKickTM Max Autosampler combines precision with performance in a benchtop flow cytometer that is configurable with up to four lasers and 16 parameters of detection. · Transform your research--get a superior level of data fidelity at speeds of up to 1 mL/min; discover rare cells and analyze more cells in a shorter period of time · Six fluorescence channels off the violet laser-- expand your capabilities in multicolor flow cytometry · Simplified sample prep--no-wash, no-lyse sample prep options streamline your workflow · Flexibility--convert between tubes and plates with a simple click of the mouse · Option for automation--designed for walk-away performance with clog-resistant fluidics and robust data analysis software · Compatible--mammalian cells, algae, bacteria, yeast, parasites, and plant cells successfully analyzed Table 10. Attune NxT Flow Cytometer specifications. Attribute Optics Fluidics Performance Specification · Laser wavelength (nm): Violet 405, blue 488, green 532, yellow 561, red 637 · Emission filters: Up to 14 color channels with wavelength-tuned photomultiplier tubes (PMTs); user-changeable, keyed filters · Flow cell: Quartz cuvette gel coupled to 1.2 numerical aperture (NA) collection lens, 200 x 200 m · Sample analysis volume: 20 L4 mL · Custom sample flow rates: 12.51,000 L/min · Sample delivery: Positive-displacement syringe pump for volumetric analysis · Fluorescence sensitivity: 80 molecules of equivalent soluble fluorochrome (MESF) for FITC, 30 MESF for PE, 70 MESF for APC · Fluorescence resolution: CV <3% for the singlet peak of propidium iodidestained chicken erythrocyte nuclei (CEN) · Data acquisition rate: Up to 35,000 events/sec, 34 parameters, based on a 10% coincidence rate per Poisson statistics · Maximum electronic speed: 65,000 events/sec with all parameters · Carryover: Single-tube format: <1% · Forward and side scatter sensitivity: Able to discriminate platelets from noise · Forward and side scatter resolution: Optimized to resolve lymphocytes, monocytes, and granulocytes in lysed whole blood · Minimum particle size: 0.2 m on side scatter using submicron bead calibration kit from Bangs Laboratories--0.1 m on side scatter under following conditions: Using an Attune NxT Flow Cytometer with standard blocking configuration, an Attune NxT 488/10 Filter (Cat. No. 100083194), and Attune Focusing Fluid (Cat. No. 4488621, 4449791, or A24904) that has been passed through a 0.025 m filter Find out more about instruments and robotics at thermofisher.com/attune 14 Application spotlight--analyze samples for CRISPR-edited cells on the Attune NxT Flow Cytometer Cell isolation and activation Cell editing Functional KO analysis Genomic analysis Dynabeads Untouched Human CD4 T Cells Kit Neon Transfection System TrueCut Cas9 Protein v2 TrueGuide Synthetic gRNA Fluorescent dyes eBioscience antibodies Attune NxT Flow Cytometer GeneStudio S5 System 3500 Genetic Analyzer Geonomic Cleavage Detection Kit SSC-A :: SSC-A SSC-A :: SSC-A SSC-A :: SSC-A Knockout efficiency (%) A 1.0M 800K 600K 400K 200K 0 Untransfected sample TCR-- 11.2% TCR+ 88.2% YL1-A :: YL1-A TRAC T1 gRNA + Cas9 RNP 1.0M 800K TCR-- 93.4% TCR+ 6.31% 600K 400K 200K 0 YL1-A :: YL1-A TRBC T1 gRNA + Cas9 RNP 1.0M 800K TCR-- 81.8% TCR+ 17.8% 600K 400K 200K 0 YL1-A :: YL1-A B 120 100 80 60 40 20 0 TRAC T1 TRAC T2 TRBC T1 TRBC T2 Figure 16. High-efficiency functional knockout in T cells. T cells were isolated from PBMCs (from a healthy donor) using Dynabeads magnetic beads, and then transfected with InvitrogenTM TrueCutTM Cas9 Protein v2 and InvitrogenTM TrueGuideTM Modified Synthetic sgRNAs targeting T cell receptor alpha (TRAC) or beta (TRBC) regions using the InvitrogenTM NeonTM Transfection System. (A) Analysis by flow cytometry following binding with antibody specific to the T cell receptor (TCR) shows >90% functional knockdown of the receptor. For both TRAC and TRBC, gRNAs specific for two different genomic DNA targets (T1 and T2) were tested, and results are shown only for the T1 target in each case. (B) Summary of NGS-based analysis of cleavage efficiency at two different genomic DNA targets (T1 and T2) for both TRAC and TRBC loci. Services and support Instrument service plans and warranties Extended-coverage service plans are available at the time of instrument purchase. With these service plans you can maximize system uptime, reduce overall repair costs, get fast repair turnaround time from a manufacturertrained and certified field service engineer (FSE), extend instrument life, and help keep it running at peak performance. Choose from a variety of service options that balance budget, productivity, uptime, and regulatory requirements. Plans start with the most basic repair models and scale to premium offerings, including advanced support and compliance services. Technical support for help with flow cytometry experiments Technical support and specialists assist with panel design and help choose the correct antibodies for your needs, including new experiments and quality control. Each specialist helps troubleshoot experiments and product performance issues, as well as designing and helping customers implement complex flow cytometry panels (>30 colors), all remotely via phone or email. Services are available globally. "Our team includes a variety of experienced professionals with an average of 14 years of research experience. While we are technically oriented, our focus is the achievement and satisfaction of our customers and that is how we measure our own success." Ricky Williams, Commercial Global Service and Support Build a personalized service quote at thermofisher.com/serviceselector 15 Ordering information Product Cell stimulation reagents Cell Stimulation Cocktail Concanavalin A (Con A) Solution (500X) Lipopolysaccharide (LPS) Solution (500X) AntiHuman CD3, Functional-Grade Purified (clone OKT3) AntiHuman CD28, Functional-Grade Purified (clone CD28.2) Macrophage Colony-Stimulating Factor (M-CSF) Flow cytometry antibodies eBioscience flow cytometry antibodies Fixatives eBioscience Flow Cytometry Staining Buffer FIX & PERM Cell Permeabilization Kit eBioscience Intracellular Fixation and Permeabilization Buffer Set eBioscience Foxp3/Transcription Buffer Set Viability dyes CellTrace Blue Cell Proliferation Kit, for flow cytometry CellTrace CFSE Cell Proliferation Kit, for flow cytometry CellTrace Far Red Cell Proliferation Kit, for flow cytometry CellTrace Violet Cell Proliferation Kit, for flow cytometry CellTrace Yellow Cell Proliferation Kit, for flow cytometry LIVE/DEAD Fixable Blue Stain LIVE/DEAD Fixable Violet Stain LIVE/DEAD Fixable Aqua Stain LIVE/DEAD Fixable Yellow Stain LIVE/DEAD Fixable Green Stain LIVE/DEAD Fixable Red Stain LIVE/DEAD Fixable Far Red Stain LIVE/DEAD Fixable Near-IR Stain Bead controls UltraComp eBeads Compensation Beads UltraComp eBeads Plus Compensation Beads AbC Total Antibody Compensation Bead Kit ArC Amine Reactive Compensation Bead Kit GFP BrightComp eBeads Compensation Beads CountBright Plus Absolute Counting Beads AccuCheck Counting Beads AccuCheck ERF Reference Particles LIVE/DEAD BacLight Bacterial Viability and Counting Kit Instruments Attune NxT Flow Cytometer CytKick Autosampler CytKick Max Autosampler Cat. No. 00-4970-93 00-4978 00-4976 16-0037 16-0289 PHC9504 thermofisher.com/flowantibodies 00-4222-57 GAS003 88-8824-00 00-5523-00 C34568 C34570 C34564 C34557 C34567 L23105 L34955 L34957 L34959 L23101 L23102 L10120 L10119 01-2222-41 01-3333-42 A10497 A10346 A10514 C36995 PCB100 A55950 L34856 thermofisher.com/attune A42901 A42973 Download guide Find out more at thermofisher.com/flow For Research Use Only. Not for use in diagnostic procedures. Not for resale. Super Bright Polymer Dyes are sold under license from Becton, Dickinson and Company. © 2019, 2021 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Cy is a registered trademark of GE Healthcare. Cytek, Aurora, and SpectroFlo are trademarks of Cytek Biosciences. DRAQ7 is a trademark of BioStatus Limited. Excel is a trademark of Microsoft Corp. COL33963 0121Adobe InDesign 16.0 (Macintosh) Adobe PDF Library 15.0