Singapore (English)
-
Your selected country is
Singapore
- Change country/language
Products
-
Reagents
- Flow Cytometry Reagents
-
Western Blotting and Molecular Reagents
- Immunoassay Reagents
-
Single-Cell Multiomics Reagents
- BD® AbSeq Assay
- BD Rhapsody™ Accessory Kits
- BD® Single-Cell Multiplexing Kit
- BD Rhapsody™ Targeted mRNA Kits
- BD Rhapsody™ TCR/BCR Profiling Assays for Human and Mouse
- BD Rhapsody™ Whole Transcriptome Analysis (WTA) Amplification Kit
- BD® OMICS-Guard Sample Preservation Buffer
- BD Rhapsody™ ATAC-Seq Assays
-
Functional Assays
-
Microscopy and Imaging Reagents
-
Cell Preparation and Separation Reagents
Discover & Learn
-
Training
- Flow Cytometry Basic Training
-
Product-Based Training
- FACSAria Product Based Training
- FACSMelody Product-Based Training
- FACSLyric Product-Based Training
- FACSCanto Product-Based Training
- LSRFortessa Product-Based Training
- FACSymphony Product-Based Training
- FACSDuet Product-Based Training
- HTS Product-Based Training
- BD FACSDiscover™ S8 Cell Sorter Product Training
-
Advanced Training
-
Thought Leadership
-
Product News
- Blogs
- Scientific Publications
-
Events
- Nature Research Academies Workshop 2023
- CYTO 2023: Advancing the World of Cytometry
- Singapore Gene & Cell Therapy Conference 2023
- EuroFlow Educational Workshop
- Nature Research Masterclass 2023
- Novel Approaches to Single-Cell Plant Research: from Real-Time Imaging Cell Sorting to Single-Nuclei Transcriptomics
- Advances in Immune Monitoring Series
-
Product News
Resources & Tools
Support
-
- BD® AbSeq Assay
- BD Rhapsody™ Accessory Kits
- BD® Single-Cell Multiplexing Kit
- BD Rhapsody™ Targeted mRNA Kits
- BD Rhapsody™ TCR/BCR Profiling Assays for Human and Mouse
- BD Rhapsody™ Whole Transcriptome Analysis (WTA) Amplification Kit
- BD® OMICS-Guard Sample Preservation Buffer
- BD Rhapsody™ ATAC-Seq Assays
-
- FACSAria Product Based Training
- FACSMelody Product-Based Training
- FACSLyric Product-Based Training
- FACSCanto Product-Based Training
- LSRFortessa Product-Based Training
- FACSymphony Product-Based Training
- FACSDuet Product-Based Training
- HTS Product-Based Training
- BD FACSDiscover™ S8 Cell Sorter Product Training
-
- Nature Research Academies Workshop 2023
- CYTO 2023: Advancing the World of Cytometry
- Singapore Gene & Cell Therapy Conference 2023
- EuroFlow Educational Workshop
- Nature Research Masterclass 2023
- Novel Approaches to Single-Cell Plant Research: from Real-Time Imaging Cell Sorting to Single-Nuclei Transcriptomics
- Advances in Immune Monitoring Series
- Singapore (English)
- Change country/language
You are now leaving the BD Biosciences website. The site you are about to visit is operated by a third party. The link to this site neither makes nor implies any representation or warranty for any products or services offered on a third-party site and is intended only to enable convenient access to the third-party site and for no other purpose. Do you want to continue?
Old Browser
For the best web browsing experience, please use Chrome, Safari or Firefox, minimum versions 77.0.3865, 12.1.2 and 68, respectively.
Please Note
This page has been recently translated and is available in French now.
Please confirm your location
Looks like you're visiting us from {countryName}.
Would you like to stay on the current country site or be switched to your country?
Cell Surface
Cell Surface Staining of Human PBMCs and Suspension Cell Lines
Reagents and Materials Required
Full Name | Short Name | Catalog Number |
BD Pharmingen™ Stain Buffer with BSA or BD Pharmingen™ Stain Buffer with FBS | Stain Buffer | 554657 or 554656 |
Microwell plates (round bottom wells) or 12 x 75-mm polypropylene round bottom tubes | plates or tubes | |
Fluorescent antibodies | antibodies | |
Centrifuge |
Optional Reagents
Full Name | Short Name | Catalog Number |
Human BD Fc Block™ | Fc Block | 564219/564220 |
Labeled secondary antibodies | secondary antibodies | |
Labeled streptavidin conjugates | streptavidin conjugates | |
BD Cytofix™ buffer | BD Cytofix buffer | 554655 |
Procedural Notes
- For peripheral blood mononuclear cell (PBMC) preparation, use the manufacturer's directions for Ficoll products.
- For most applications, BSA as a blocking agent is sufficient, but investigators may use FBS if more stringent blocking is required.
- No wash is needed prior to antibody staining.
- Determination of optimal antibody concentration may be necessary. For test size antibody products, add the recommended test size volume. Staining time may be increased (>45 minutes) depending on the avidity of the fluorescent antibody.
- If analysis must be delayed, then the stained cells can be fixed with buffered paraformaldehyde (for example, BD Cytofix buffer, see the product TDS for a detailed protocol) for 30 minutes at 4°C, washed, resuspended in Stain Buffer, and then stored at 4°C (protected from light). The fixed cells should be analyzed as soon as possible. We have not tested all fluorescently conjugated antibodies for this fixation. Therefore, researchers may need to verify if this fixation will affect antibody binding and fluorescence intensity.
Procedure
- If necessary, resuspend PBMCs or cell lines with Stain Buffer.
- Wash the cells twice in cold Stain Buffer and pellet the cells by centrifugation at 300 g at 4°C.
- Resuspend the cell pellet with cold Stain Buffer to a final concentration of 2 x 107 cells/mL.
- Distribute 100-µL aliquots of the cell suspension (106 cells) to either tubes or the round-bottomed wells of plates.
- (Optional) To block non-specific Fc-mediated interaction, add 2.5 μg of Fc Block per 106 PBMCs per tube (or per well) and incubate for 10 minutes at room temperature.
- Add antibodies to cells and incubate for 20 minutes on ice, protected from light.
- Wash the cells two times with either 200-µL (for plates) or 1-mL (for tubes) volumes of Stain Buffer. Centrifuge cells at 300 g for 5 minutes.
- Carefully aspirate (for microwell plates or tubes) or invert and blot away (for tubes) supernatants from cell pellets.
- Tap tubes/plates to loosen the cell pellet.
- For indirect immunofluorescent staining of cells, repeat steps 5 and 6 with either a secondary antibody or a streptavidin conjugate in 100 µL of Stain Buffer.
- Resuspend the cell pellet in either 200-µL (for microwell plates) or 0.5-mL (for tubes) volumes of Stain Buffer.
- Analyze stained cell samples by flow cytometry.
Report a Site Issue
This form is intended to help us improve our website experience. For other support, please visit our Contact Us page.
Successfully submitted.