Leinco Technologies carefully formulated this product at a neutral pH (pH 7.0 - 7.4) (Test at 1X concentration) so that when you dilute to a working volume it is ready to use (Note: Some pH Adjustment may be required).
This product is a buffered salt solution that is supplemented with 0.5% bovine serum albumin (BSA) proteins. Prior to packaging this product 0.2 μm filtered into non-sterile containers. More detailed information regarding formulation can be requested from technical support.
Storage and Handling
Store at 2-8°C. Do Not Freeze
This FCM or BSA Stain Buffer can be used for the immunofluorescent staining of single-cell suspensions prepared from either lymphoid tissues, bone marrow, peripheral blood, or cultured cells. This buffer is also useful for applications using fluorescent reagents as well as for the suspension, washing, and storage of cells destined for flow cytometric analysis (or fluorescence microscopy).
Application: FCM Stain Buffer (BSA) is useful for staining cells with biotinylated antibodies and fluorochrome- conjugated avidins because this staining medium contains no biotin. When present in staining media, free biotin can interfere with the binding of fluorescent-avidins to biotinylated antibodies that have complexed with their cognate, cell-associated antigens. Moreover, free biotin can bind to cells and contribute to an increase in the non-specific, background staining of cells that are exposed to fluorochrome-conjugated avidins. As such, FCM Stain Buffer (BSA) is designed to maintain cell viability and maximize fluorescence signal intensities generated by pH-sensitive fluorochromes, such as FITC. The FCM Stain Buffer (BSA) should be used at a 1X concentration.
Directions for Use
1. Prepare single-cell suspensions from either lymphoid tissue, bone marrow, peripheral blood or cell cultures using standard protocols.
2. Wash the cells twice in cold 1X FCM Stain Buffer (BSA). Pellet the cells by centrifugation. Resuspend the cell pellet with cold Leinco FCM Stain Buffer (BSA) to a final concentration of 2 x 10e7 cells/ml.
3. Distribute 50 μl aliquots of the cell suspension to either tubes or the round-bottomed wells of microwell plates. Be sure to label all tubes clearly.
4. Dilute fluorescent antibodies to their optimal concentrations add aliquots (e.g., 20 μl) of the diluted antibodies to the tubes or microwells that contain the target cell suspensions. Incubate for 20 minutes on ice & protect from light.
5. Wash the cells two times with either 200 μl (for microwell plates) or 1 ml (for tubes) volumes of Stain Buffer (BSA) to remove unbound antibodies. Centrifuge cells as 300 x g for 5 min. After each centrifugation, carefully aspirate (for microwell plates or tubes) or invert and blot away (for tubes) supernatants from cell pellets.
6. Resuspend the cell pellet in either 200 μl (for microwell plates) or 0.5 ml (for tubes) volumes of Leinco FCM Stain Buffer (BSA). Transfer stained cells from microwell plates to the appropriate tubes for flow cytometric analysis (adjust final volume to ~0.5 ml).
7. Analyze stained cell samples either by flow cytometry or by fluorescence microscopy as soon as possible (e.g., ≤ 2-3 hours) after staining. If analysis must be delayed, then the stained cells can be fixed and stored at 4°C (protected from light).
This product is manufactured under optimal conditions in class 10,000 cleanrooms. Leinco Technologies can provide the client with a copy of C of A if requested.
Products are for research use only. Not for use in diagnostic or therapeutic procedures.