BD CaliBRITE beads are designed for use with FACSComp or AutoCOMP software and the FACS family of flow cytometers (FACSCalibur, FACSort, FACScan. values for BD Calibrite beads. To edit, see page A Target file is also created for. HLA-B Although used by. BD FACSComp software, the file is not editable. Product Name: BD CALIBRITE BEADS. Synonyms: BD CALIBRITE BEADS; CALIBRITE BEADS. CAS: MF: MW: 0. EINECS: Mol File: Mol File. BD CALIBRITE .
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Documents Flashcards Grammar checker. Wellington, Auckland, New Zealand bdbiosciences. The beads are used to adjust instrument settings, set fluorescence compensation, and check instrument sensitivity. Daily use is recommended for monitoring instrument performance over time. BD Calibrite beads are for in vitro diagnostic use.
Bedas 2color kit contains three different types of BD Calibrite beads: The 3-color kit contains these beads plus a PerCPlabeled bead. An APC-labeled bead is available separately and may be used with the 3-color kit to perform four-color setup. This instructions for use IFU provides information for two- three- and four-color setup. Refer to the information appropriate to the instrument setup you are performing.
The flow cytometer has separate detectors or photomultiplier tubes PMTs that detect light signals.
Both scatter and fluorescent light signals are detected. Because BD Calibrite beads simulate unstained cells and cells that have been stained labeled with fluorochrome-conjugated antibodies, the beads are used to adjust the instrument settings before cell bsads are run on the flow cytometer.
The following list illustrates PMT light signal detection: BD Calibrite beads are used to determine the appropriate compensation settings. After the instrument settings have been determined, BD Calibrite beads are used to evaluate instrument sensitivity. Forward scatter FSC and side scatter SSC instrument sensitivity are measured by the mean channel separation between the light-scatter signal of the beads and background signal electronic and optical. FL1, FL2, and FL3 fluorescence sensitivity is determined by measuring the mean channel separation between the signal of the labeled beads and the unlabeled beads.
A minimum channel separation must be met for the scatter and fluorescence parameters. This allows cells to be distinguished from sample debris or background signal and for dimly stained cells to be distinguished from unstained cells.
This product is licensed for sale only for in vitro diagnostics. It is not licensed for any other use. If you require any additional license to use this product and do not have one, return this material, unopened, to BD Biosciences, Qume Drive, San Jose, CAand any money paid for the material will be refunded. The FSC threshold is adjusted to a level that minimizes background signal if any.
Next, the software adjusts fluorescence compensation using a mixed-bead suspension containing equal amounts of the appropriate BD Calibrite beads.
Compensation adjustments for FL1, FL2, and FL3 correct for spectral overlap by shifting the labeled bead populations so they are aligned with the corresponding unlabeled bead populations. Following PMT and compensation adjustment, the software performs a Sensitivity Test using the appropriate mixed-bead suspension. See Optimization and Quality Control on page 4.
BD Calibrite™ Beads
Figure 1 through Figure 4 show examples of optimization for two- three- and four-color applications. All forms are provided in stabilized, buffered saline with 0.
Reagents are sufficient to perform 25 tests. One bottle is valibrite to perform 25 tests. Do not use after the expiration date shown on the label.
Do not dilute PerCP-Cy5. Beads used beyond their stability begin to show a decrease in separation between unlabeled and labeled populations, possibly resulting in Sensitivity Test failure. If deterioration is suspected, prepare a new bead suspension and check veads conditions. Concentration values are listed in the following table: For information on use, refer to the appropriate instrument manual.
See examples in Optimization and Quality Control on page 4.
BD Calibrite PerCP-Cy5.5 Beads
Preparation of Test Neads Prepare all bead suspensions immediately prior to use. Mix bead vials by gentle inversion or very gentle vortexing prior to use. Label two 12 x mm polystyrene tubes Tube A and Tube B. Gently mix the BD Calibrite bead vials, then add 1 drop of beads to each tube as indicated in the table below.
BD Calibrite™ Beads
NOTE Invert bead vials completely when adding a drop to the tube. Make sure to obtain a full drop of beads. The drop should be cloudy, indicating the beads are properly mixed. Adjust fluorescence compensation using Tube B. Perform a Sensitivity Test using Tube B. Generate a printout of the Sensitivity Test results and keep the printouts in a log book. Record PMT voltages and channel separations obtained for each parameter in a daily log sheet.
Optimize settings for your sample, as needed. Instrument settings might need to be manually optimized before running cells.
Optimization and Quality Control Because leucocytes have different optical properties than BD Calibrite beads, optimization of instrument settings with cell samples is important. Prepare a blood sample daily from a normal donor. Use the same staining method and run in parallel with the test samples. Optimize instrument settings following two-color setup using a blood sample stained with any combination of monoclonal antibodies that identifies separate non-overlapping cell populations, such as FITClabeled and PE-labeled monoclonal antibodies.
Optimization following three- and four-color setup can vary depending on the application. See Figure 1 through Figure 5 for examples. Always refer to the appropriate application note or reagent IFU. NOTE Different immunophenotyping preparation methods might require different optimization procedures. It might be necessary to adjust the FSC and SSC amplifiers so that all leucocyte populations are on scale, and to adjust compensation and threshold settings see Figure 1.
BD CALIBRITE BEADS
Optimizing Scatter Figure 1 shows a lysed whole blood LWB sample from a normal donor before and after optimization. Notice populations with a lower FSC signal than lymphocytes debris, for example can be excluded by increasing the FSC threshold level.
Adjustment is similar for PerCP-Cy5.
The light scatter sensitivity is determined by the amount of channel separation between the mixed bead population and instrument background signal. The channel separation and PMT voltages for each of the four parameters should be maintained in a daily log to track instrument performance.
NOTE Over a period of time, the fluorescence separation might decrease. The decrease in separation for a wide variety of bead lots has been within 2. Corrective action might be required if the average separation varies by more than 2. Observations of greater variations on a single instrument can be indicative of instrument instability. In some cases the software may not be able to automatically set up the instrument. If this occurs, manually adjust the settings.
Beads used beyond their stability begin to show a decrease in separation between unlabeled and labeled populations, resulting in Sensitivity Test failure.
The suspensions are stable for a longer period of time in Bead Dilution Buffer. For further assistance, contact your BD Biosciences service representative. UV Bead lab with graph. Weather and Climate for Educators.
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