BD Stabilizing Fixative
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1 BD Stabilizing Fixative 04/ IVD 3X concentrate Catalog No BD, BD Logo and all other trademarks are property of Becton, Dickinson and Company BD Becton, Dickinson and Company BD Biosciences 2350 Qume Drive San Jose, CA USA Benex Limited Pottery Road, Dun Laoghaire, Co. Dublin, Ireland Tel Fax BD Biosciences European Customer Support Tel Fax Becton Dickinson Pty Ltd, 4 Research Park Drive, Macquarie University Research Park, North Ryde NSW 2113, Australia Becton Dickinson Limited, 8 Pacific Rise, Mt. Wellington, Auckland, New Zealand bdbiosciences.com ClinicalApplications@bd.com 1. INTENDED USE BD stabilizing (3X concentrate) is intended for preserving immunofluorescence staining of human peripheral blood cells with monoclonal antibodies prior to flow cytometric analysis. 2. SUMMARY AND EXPLANATION Fluorochromes requiring efficient energy transfer to Cy 7 * (PE-Cy7 and APC-Cy7 ) and H7 (APC-H7), can break down and begin to show emission from the donor part of the fluorochrome. This breakdown can be caused by exposure to light during the staining process, and by prolonged storage in formaldehydecontaining buffers after staining. decreases the rate of this second type of breakdown in these tandem conjugates. 3. PRINCIPLES OF THE PROCEDURE When using a lyse/wash (LW) staining procedure for Cy7 and H7 tandem conjugates, resuspend the washed cells in 1X rather than 1% paraformaldehyde (PFA) solution. * Cy is a trademark of GE Healthcare. This product is subject to proprietary rights of GE Healthcare and Carnegie Mellon University, and is made and sold under license from GE Healthcare. 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, 2350 Qume Drive, San Jose, CA 95131, and any money paid for the material will be refunded. APC-Cy7: US patent 5,714,386 1
2 4. REAGENT Reagent provided (3X concentrate) is provided as 90 ml of a proprietary buffered solution containing less than 3% formaldehyde. This quantity is sufficient for 500 tests when used in BD LW procedures. is delivered as a 3X concentrate for use on the BD FACS Lyse Wash Assistant (LWA). Reagent contains 2.81% formaldehyde, CAS number , and 4.01% disodium tetraborate decahydrate, CAS number Danger H341 Suspected of causing genetic defects. H350 May cause cancer. Route of exposure: Inhalative. H360 May damage fertility or the unborn child. H317 May cause an allergic skin reaction. Wear protective gloves / eye protection. Wear protective clothing. IF ON SKIN (or hair): Remove/Take off immediately all contaminated clothing. Rinse skin with water/shower. IF exposed or concerned: Get medical advice/attention. If skin irritation or rash occurs: Get medical advice/attention. Dispose of contents/container in accordance with local/regional/national/international regulations. Precautions For In Vitro Diagnostic Use. WARNING All biological specimens and materials coming in contact with them are considered biohazards. Handle as if capable of transmitting infection 1,2 and dispose of with proper precautions in accordance with federal, state, and local regulations. Never pipette by mouth. Wear suitable protective clothing, eyewear, and gloves. Dilution instructions Dilute 1 part of the 3X concentrate with 2 parts of room temperature (20 C 25 C) deionized (DI) water. The prepared 1X solution is stable for one month when stored in a polystyrene container at 2 C 8 C. WARNING Do not store 1X in high-density polyethylene (HDPE) bottles because performance can be affected. Storage and handling (3X) is stable until the expiration date shown on the bottle label when stored as directed. Do not use the reagent if you observe any change in appearance. Precipitation or discoloration indicates instability or deterioration. WARNING Do not store 3X in LWA tanks for more than five months. Although LWA tanks are made of HDPE, can be stored for up to five months if the tanks are pre-washed with DI water. WARNING Do not store samples stained with AmCyan conjugates in. Exposure of these conjugates to can cause increased into the FITC channel. WARNING Do not store samples stained with PE conjugates in because this can cause a decrease in staining intensity. 2
3 5. INSTRUMENT Samples fixed with can be analyzed by flow cytometers equipped with appropriate computer hardware and software. The flow cytometer must be equipped to detect light scatter and the appropriate fluorescence, and be equipped with appropriate analysis software (such as BD CellQuest, BD CellQuest Pro, BD FACSDiva, or BD FACSCanto clinical software) for data acquisition and analysis. See the cytometer user s guide for instructions. 6. PROCEDURE Reagent provided, 3X concentrate (Catalog No ) Reagents and materials required but not provided 1X BD FACS lysing solution. For dilution instructions and warnings, see the product instructions for use (IFU). Falcon disposable 12 x 75-mm polystyrene test tubes or equivalent BD Biosciences monoclonal antibodies to human leucocyte antigens Vortex mixer Centrifuge Micropipettor with tips BD CellWASH (Catalog No ) or a wash buffer of phosphate buffered saline (PBS) with 0.1% sodium azide Stain buffer solution consisting of PBS plus 0.2% bovine serum albumin (BSA) with 0.1% sodium azide Falcon is a registered trademark of Corning Incorporated. 1X. See Dilution instructions on page 2. Preparing samples Stain whole blood specimens following the instructions in the appropriate reagent IFU. Lyse red blood cells as directed using diluted (1X) BD FACS lysing solution. Use care to protect the tubes from direct light. Perform the procedure at room temperature (20 C 25 C). 1. Add the appropriate volume of fluorochrome-conjugated monoclonal antibody to 100 µl of whole blood in a 12 x 75-mm polystyrene test tube. See the antibody vial label for volume. 2. Vortex gently and incubate for 15 to 30 minutes in the dark at room temperature (20 C 25 C). 3. Add 2 ml of 1X BD FACS lysing solution to each tube. 4. Vortex the tubes gently and incubate for 10 minutes in the dark at room temperature. 5. Centrifuge the tubes at 300g for 5 minutes. 6. Aspirate the supernatant. 7. Add 2 ml of BD CellWASH solution or stain buffer to each tube. See the fluorochrome-conjugated monoclonal antibody IFU for more information. 8. Centrifuge the tubes at 300g for 5 minutes. 9. Aspirate the supernatant. 10. Add 0.5 ml of 1X to the tubes. CAUTION Do not use for multicolor analysis using a PE or AmCyan conjugate. See the 3
4 fluorochrome-conjugated monoclonal antibody IFU for more information. 11. Vortex the tubes gently and incubate for 30 minutes in the dark at 2 C 8 C. 12. Mix the tubes thoroughly before analysis. Store the tubes at 2 C 8 C in the dark until analyzed. We recommend analyzing within 24 hours of staining. CAUTION Some PE-Cy7, APC-Cy7, and APC-H7 conjugates show changes in their emission spectra with prolonged exposure to paraformaldehyde. For overnight storage of stained cells, wash and resuspend in buffer without paraformaldehyde after 1 hour of fixation. 7. RESULTS The following representative data were obtained with peripheral blood samples treated as instructed in this IFU. Final resuspension was in PBS, PBS with 1% paraformaldehyde, or. Table 1 Percent shown at 2 hours, on CD45 APC-Cy7 48 h and PBS PFA Table 2 Percent shown at 2 hours, on CD20 APC-Cy7 48 h and PBS PFA Table 3 Percent shown at 2 hours, on CD8 APC-Cy7 48 h and PBS PFA NOTE Storing stained cells in can result in a slight degradation of forward scatter performance. 8. LIMITATIONS is specifically formulated for use with BD FACS brand flow cytometers. EDTA is the anticoagulant of choice. BD has limited information concerning use of other anticoagulants, such as heparin. WARRANTY Unless otherwise indicated in any applicable BD general conditions of sale for non-us customers, the following warranty applies to the purchase 4
5 of these products. THE PRODUCTS SOLD HEREUNDER ARE WARRANTED ONLY TO CONFORM TO THE QUANTITY AND CONTENTS STATED ON THE LABEL OR IN THE PRODUCT LABELING AT THE TIME OF DELIVERY TO THE CUSTOMER. BD DISCLAIMS HEREBY ALL OTHER WARRANTIES, EXPRESSED OR IMPLIED, INCLUDING WARRANTIES OF MERCHANTABILITY AND FITNESS FOR ANY PARTICULAR PURPOSE AND NONINFRINGEMENT. BD S SOLE LIABILITY IS LIMITED TO EITHER REPLACEMENT OF THE PRODUCTS OR REFUND OF THE PURCHASE PRICE. BD IS NOT LIABLE FOR PROPERTY DAMAGE OR ANY INCIDENTAL OR CONSEQUENTIAL DAMAGES, INCLUDING PERSONAL INJURY, OR ECONOMIC LOSS, CAUSED BY THE PRODUCT. REFERENCES 1. Protection of Laboratory Workers from Occupationally Acquired Infections; Approved Guideline Third Edition. Wayne, PA: Clinical and Laboratory Standards Institute; CLSI document M29-A3. 2. Centers for Disease Control. Perspectives in disease prevention and health promotion update: universal precautions for prevention of transmission of human immunodeficiency virus, hepatitis B virus, and other bloodborne pathogens in health-care settings. MMWR. 1988;37:
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