Description
BD FACS™ Pre-Sort Buffer was designed to aid in preparing and maintaining single cell suspensions prepared from lymphoid tissues, bone marrow, peripheral blood, cultured cells (including pluripotent stem cells) or other sources. Additives may be necessary for sensitive cell types requiring specific nutrients. The Pre-Sort Buffer contains no phenol red and is an optically clear buffer to help minimize background. The buffer has been formulated to minimize cell clumping which facilitates consistent drop formation on fluorescence activated cell sorters. The buffer has minimal calcium and magnesium to minimize cell aggregation as these cations are necessary cofactors for many cell adhesion molecules. The Pre-Sort Buffer contains an FBS-based protein to help maintain cells in a viable state during cell sorting applications but the buffer does not contain EDTA, which can have deleterious effects on cells. It is useful for the dilution and application of fluorescent reagents as well as for the suspension, washing, and storage of cells destined for florescence activated cell sorting (FACS) or flow cytometric analysis.
Preparation And Storage
Thaw completely at 4°C before first use. Distribute 25 ml aliquots (or desired single use volumes) of complete buffer into sterile containers and store at -20°C. The complete buffer is stable at -20°C for 6 months. Each aliquot should be considered single use and used completely on the day of thaw to ensure optimal activity (avoid multiple freeze/thaw cycles).
Recommended Assay Procedures
Flow Cytometry (Direct immunofluorescence staining):
1. Thaw your BD FACS™ Pre-Sort Buffer aliquot completely at 4°C.
2. Prepare single-cell suspensions from tissue, bone marrow, peripheral blood or cell cultures using standard protocols.
3. Wash the cells twice in cold complete Pre-Sort Buffer. Resuspend the cell pellet with cold Pre-Sort Buffer to a final concentration of ~1 x 10e7 cells/ml.
a. If necessary dilute fluorescent antibodies to their predetermined optimal concentrations in complete Pre-Sort Buffer.
4. Add appropriate amount of conjugated antibody to the cell suspension. Incubate for 20 minutes on ice protected from light.
a. Staining time may be increased (≥ 45 min) depending on the avidity of the fluorescently conjugated antibody.
5. Wash the cells two times with complete Pre-Sort Buffer. Centrifuge cells at 300 x g for 5 min. After each centrifugation, carefully aspirate or invert and blot away supernatants from cell pellets.
6. Resuspend the cell pellet in complete Pre-Sort Buffer and transfer stained cells to the appropriate tubes for flow cytometric analysis and/or sorting (e.g. adjust final volume to provide ~5 x 10e6 cells/ml).
7. Immediately sort cells based on an optimized sort protocol.
Note 1: DNase II can be added to Pre-Sort Buffer to help discourage clumping of cell suspensions that have a high amount of cell death. The presence of free DNA in cell suspension can contribute to cell clumping. DNase I is not compatible with the Pre-Sort Buffer due to the necessity of cations for DNase I to function. DNase II contains levels of endotoxin and may vary by batch. Depending on your application, you may wish to add DNase II to the Pre-Sort Buffer. However, you may also need to determine if the levels of endotoxin are appropriate for your application.
Note 2: Pre-Sort Buffer does not contain antibiotics. Antibiotics can be added to the Pre-Sort Buffer as appropriate.
Note 3: Pre-Sort Buffer can similarly be used for the indirect immunofluorescent staining of cells. In this case, repeat steps 4 and 5 with second step reagents when using either unlabeled or biotinylated primary antibodies.
Note 4: Pre-Sort Buffer can also be used for the immunofluorescent staining of surface antigens expressed by cells that are destined to be fixed and immunofluorescently stained for intracellular antigens such as cytokines and transcription factors. Cells stained for intracellular antigens can be resuspended and maintained (i.e., at 4°C, protected from light) in Pre-Sort Buffer prior to analysis by flow cytometry.
Note 5: Pre-Sort Buffer is not a post-sort collection buffer. Please collect cells in buffer/media that is appropriate for downstream applications.
Note 6: Pre-Sort Buffer is compatible with commonly used viability dyes such as 7-AAD.
Product Notices
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- Alexa Fluor® is a registered trademark of Molecular Probes, Inc., Eugene, OR.
- Accutase is a registered trademark of Innovative Cell Technologies, Inc.
- Ficoll-Paque is a trademark of Amersham Biosciences Limited.
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
Companion Products
Development References (1)
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McIntyre CA, Renin G. Reduction in Endotoxin Levels After Performing the Prepare for Aseptic Sort Procedure on the BD FACSAria II Flow Cytometer. Available: http://www.bdbiosciences.com/documents/FacsariaII_Endotoxin.pdf 2015, February 9.
Please refer to Support Documents for Quality Certificates
Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described
Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims. Comparisons are not made against non-BD technologies, unless otherwise noted.
