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View additional product information for AbC™ Total Antibody Compensation Bead Kit - FAQs (A10513, A10497)
14 product FAQs found
Check the isotype of the two antibodies to see if they are different. If so, we offer isotype-specific goat anti-mouse secondary antibodies that can be used together with minimal cross-reactivity. If the two mouse antibodies are of the same isotype, then you would need to make direct conjugates, such as with our antibody labeling kits or APEX, and SiteClick kits.
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Here are some tips to follow:
- Make sure that the beads were never frozen.
- Avoid mixing by vortexing too long (no longer than 10 seconds) or sonication as this may promote denaturation of the protein on the surface of the beads.
- Use the product within the warranty period (one year).
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Yes, the AbC Total Antibody Compensation Bead Kit can bind Fab dimers as long as the Fab dimers are derived from either mouse, rat, hamster, or rabbit species.
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As an alternative, you may covalently attach your antibody to colorless, 5 µm Aldehyde/Sulfate Latex Beads (Cat. No. A37306). The aldehyde moiety on the bead surface directly binds with primary amines on the surface of the antibodies in simple buffers such as PBS (pH 7.4). Make sure that the buffer does not contain any primary amines (i.e., do not use Tris or glycine buffers). To avoid making bead-antibody-bead-antibody multimers, add excess antibody relative to the beads.
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It is possible. But if you have a lot of values over 100, you probably need to look at the voltages that you are using for your data collection.
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By running single-color controls, it is possible to remove signal of one fluorophore that spills over into the collection channel for another fluorophore.
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For compensation, you need to prepare a singly stained sample (or compensation beads) for each color parameter that you are using. In addition, we recommend that you use FMO (flow minus one) controls. These are controls in which you label cells or beads with every color in your panel, omitting one. Make one FMO control for each color. These controls are important for helping you properly set gates on your data.
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In a perfect world, the fluorescence emission profile for each individual fluorophore would be a very intense, narrow peak, well separated from all other emission peaks. In reality, organic dyes and fluorescent proteins have broad emission peaks, and compensation must be employed (during or after data acquisition) to correctly assign fluorescence signal to each fluorophore. Compensation is important because it removes fluorescent signal from overlapping spectra so you know that the signal you see is only the signal from the fluorophore of interest.
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All that you need for compensation controls is an unstained sample of your cells (or negative control beads) and samples with each of your fluorophores - one per tube. You want the single-stained controls to be as bright as you expect your brightest sample to be. Antibodies can be bound to beads instead of cells using our AbC Total Antibody Compensation Bead Kit (Cat. Nos. A10513 and A10497).
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The smallest size that you can detect with the Attune NxT Acoustic Focusing Cytometer is 0.5 µm.
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The Attune NxT Autosampler, an optional accessory for the Attune NxT Acoustic Focusing Cytometer, enables rapid processing of up to 384 samples. It has broad compatibility with different plate formats, both 96- and 384-well plates. It has an intelligent probe designed to minimize clogging and carryover (<0.5%) and to prevent damage to the instrument. It mixes by aspiration rather than shaking to ensure homogeneity of the sample and maintain cell viability. Is performs automated cleaning as part of the shutdown process of the Attune NxT Cytometer. It provides consistent data regardless of sampling method (tube vs. plate) and collection rate.
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-Modular design - Multiple configurations available - field upgradable.
-Save time - 10X faster speeds with no loss in data quality.
-Simplified sample prep - No wash, no lyse options, non-clogging fluidics.
-Enables unique applications - Complex protocols on a broad range of cell types and samples.
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With the option to be configured with up to 4 lasers and 14 colors for multi-parameter analysis the Attune NxT Acoustic Focusing Cytometer was designed as a modular system to fit most experimental needs and lab budgets. The novel design of the optical path helps ensure precise fixed alignment of four spatially separated lasers onto the sample stream enabling consistency in data over time, superior performance, and superior reliability. The instrument can be configured with up to 4 solid-state lasers (405 nm, 488 nm, 561 nm, and 637 nm) with flat top beam profiles.
The Attune NxT Flow Cytometer's acoustic focusing uses ultrasonic radiation pressure (>
2 MHz) to transport particles into the center of the sample stream. This pre-focused stream is then injected into the sheath stream, which supplies an additional hydrodynamic pressure to the sample. The combination of these two forces- termed acoustic-assisted hydrodynamic focusing-results in a narrow core stream and uniform laser illumination, regardless of the sample input rate. In traditional cytometers that rely solely on hydrodynamic focusing, the sample core widens to accommodate the increases in flow rate, which results in less uniform laser light illumination.
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Cytometry is the measurement of physical or chemical characteristics of cells or particles. Flow cytometry measures these characteristics of cells or particles as they individually pass lasers in a flow cytometer instrument. Flow cytometry is performed on single cells, providing discrete measurements for each cell in the sample. It also provides a statistical distribution of the measured characteristics of the sample.
A flow cytometer is made up of three subsystems: fluidics, optics, and electronics. Fluidics moves the cells and introduces them for interrogation. Optics generates and collects the light signals. Electronics converts the optical signals to proportional electronic signals for computer analysis.
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