![](/etc.clientlibs/bdb-aem/clientlibs/clientlib-base/resources/icons/blankimage.png)
Products
-
Reagents
- Flow Cytometry Reagents
-
Western Blotting and Molecular Reagents
-
Flow Cytometry Reagents
- Immunoassay Reagents
- Single Cell Multiomics Reagents
-
Cell Preparation
-
Functional Assays
-
Microscopy and Imaging Reagents
- Western Blotting And Molecular Reagents
- Cell Preparation Separation Reagents
- Functional Cell Based Reagents
- Microscopy Imaging Reagents
- Single Cell Multiomics Reagents
- Single Cell Multinomics Reagents
Discover & Learn
Resources & Tools
-
Protocols
- BSB Protocol
-
Setting Compensation Multicolor Flow
-
Tissues Section Stain
-
Immunomicroscopy
-
Immunohistochemical
-
Immunofluorescence
-
Frozen Tissue
-
Parafin Sections
-
Fix Perm Kits
-
Protocol Direct Immunofluorscence Staining
-
Uses of Fc Block
-
Stain Lyse Wash
-
Stain Lyse No Wash
-
Mouse Splenocytes
-
Mouse Rat Leukocytes
-
Isotype Control
-
Indirect Staining Mononuclear Cells
-
Immunopurification
-
Human PBMCs
-
Human Whole Blood Samples
-
Escapee Phenomenon
-
Agarose Conjugates
-
Anti Phosphotyrosine Biotin Conjugates
-
Soluble Antibodies
-
Rabbit Polyclonal Antibodies
-
Monocloncal Antibodies
-
Horseradish Peroxidase
-
Certified Reagents
-
Biotinylated Antibodies
-
Agarose Conjugates X712261
-
Surface Staining
-
Platelet Activation
-
Intracellular Staining
-
Indirect Immunofluorescence
-
Mouse Ige
-
Cytokine Elisa
-
Induction Fas
-
Induction Dx2
-
Apoptosis By Treatment Staurosporine
-
Cell Death
-
Apo Brdu
-
Apo Direct
-
Human Cyclins
-
Detection Ki 67
-
Brdu Detection
-
Targeted mRNA Protocols
-
WTA Protocols
-
360040667732 Protocols
-
360023293831 AbSeq Protocols
-
360039007471 VDJ CDR3 Protocols
-
Annexin V Staining Protocol
-
Western Blotting with Horseradish Peroxidase Conjugates or Alkaline Phosphatase Conjugates
-
Tissue Preparation for Surface Antigen Staining
Support
-
Account Support
-
Account FAQs
- Account FAQ Answer 1
- Account FAQ Answer 2
- Account FAQ Answer 3
- Account FAQ Answer 4
- Account FAQ Answer 5
- Account FAQ Answer 6
- Account FAQ Answer 7
- Account FAQ Answer 8
- Account FAQ Answer 9
- Account FAQ Answer 10
- Account FAQ Answer 11
- Account FAQ Answer 12
- Account FAQ Answer 13
- Account FAQ Answer 14
- Account FAQ Answer 15
- Account FAQ Answer 16
- Account FAQ Answer 21
- Create Account
- Manage Account Settings
-
PrivacyPolicy
-
Terms and Conditions
-
Account FAQs
-
- Account FAQ Answer 1
- Account FAQ Answer 2
- Account FAQ Answer 3
- Account FAQ Answer 4
- Account FAQ Answer 5
- Account FAQ Answer 6
- Account FAQ Answer 7
- Account FAQ Answer 8
- Account FAQ Answer 9
- Account FAQ Answer 10
- Account FAQ Answer 11
- Account FAQ Answer 12
- Account FAQ Answer 13
- Account FAQ Answer 14
- Account FAQ Answer 15
- Account FAQ Answer 16
- Account FAQ Answer 21
- Korea (English)
- Korea (Korea)
-
국가 / 언어 변경
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.
BD™ Phosflow Protocols for Mouse Splenocytes or Thymocytes
Protocol I (Detergent Method)
Suggested Reagents
Full Name | Short Name | Catalog Number |
BD™ Phosflow Lyse/Fix Buffer I | Lyse/Fix Buffer I | 558049 |
BD™ Phosflow Perm/Wash Buffer I | Perm/Wash Buffer I | 557885 |
Procedural Notes
- Methods of activation vary for each phosphorylated cell signaling molecule. Select stimulators before beginning the protocol. For stimulators that have been tested, see Techniques for Phospho Protein Analysis (Phosflow Application Handbook).
- Instead of centrifugation, we recommend fixing cells (step 3) by adding 20-fold excess volume of warmed 1x Lyse/Fix Buffer I to the cell suspension.
Procedure
- Suspend thymocytes or splenocytes from whole thymus or spleen cells.
- Treat the cells with appropriate stimulators.
- Fix the cells immediately to maintain the phosphorylation state. (See Procedural Notes.)
- Incubate the cells at 37°C for 10 minutes.
- Centrifuge at 300g for 5 to 10 minutes and remove the supernatant.
- Vortex or mix to disrupt the pellet.
- Permeabilize the cells by adding 1 mL of Perm/Wash Buffer I for 1–10 x 10 6 cells and incubate for 10 minutes at room temperature.
- Centrifuge at 300g for 5 to 10 minutes and remove the supernatant.
- Wash the cells once with Perm/Wash Buffer I. Centrifuge at 300g for 5 to 10 minutes and remove the supernatant.
- Resuspend the cells in Perm/Wash Buffer I at a the final cell count of 1 x 10 7 cells/mL.
- Add 0.06 μg of BD™ Fc Block antibody (Cat. No. 553141 or 553142) for each 1 x 10 6 cells. Incubate on ice for 15 minutes.
- Aliquot 100 μL of the cell suspension (1 x 10 6 cells) to each tube and add the recommended volumes of BD Phosflow antibodies. (Phospho and cell surface antigens can be stained simultaneously.)
- Incubate the cells at room temperature for 30 minutes in the dark.
- Wash cells once with 2 mL of Perm/Wash Buffer I and resuspend the cells in the same buffer prior to flow cytometric analysis.
Protocol II and III (Mild or Harsh Alcohol Method)
Suggested Reagents
Full Name | Short Name | Catalog Number |
BD™ Phosflow Lyse/Fix Buffer I | Lyse/Fix Buffer I | 558049 |
BD™ Phosflow Perm Buffer II | Perm Buffer II | 558052* |
BD™ Phosflow Perm Buffer III | Perm Buffer III | 558050* |
BD Pharmingen™ Stain Buffer | Stain Buffer | 554656 |
*Select either Perm Buffer II or III depending on the surface markers and phosphospecific antibodies used.
Procedural Notes
- Methods of activation vary for each phosphorylated cell signaling molecule. Select stimulators before beginning the protocol. For stimulators that have been tested, see Techniques for Phospho Protein Analysis (Phosflow Application Handbook).
- Phospho and cell surface antigens can be stained simultaneously. However, if there is difficulty maintaining cell surface staining, it can be done immediately after fixation (step 4). (See BD™ Phosflow FAQs for more information.)
Procedure
- Prepare single-cell suspensions from mouse thymus or spleen in PBS with 5% FBS (no azide).
- Wash once with PBS with 5% FBS. Keep the cells at 4°C before stimulation.
- Treat the cells with appropriate stimulators.
- Fix the cells immediately to maintain the phosphorylation state by adding one volume of single-cell suspension to 20 volumes of warmed 1x Lyse/Fix Buffer.
- Incubate the cells at 37°C for 10 minutes. Centrifuge at 300g for 5 to 10 minutes and remove the supernatant.
- Vortex or mix to disrupt the pellet.
- Permeabilize the cells by adding 1 mL of Perm Buffer II or III for 1–10 x 10 6 cells and incubating for 30 minutes on ice.
- Wash the cells twice with Stain Buffer. Centrifuge at 300 g for 5 minutes and remove the supernatant.
- Resuspend the cells in Stain Buffer at a the final cell count of 1 x 10 7 cells/mL.
- Add 0.06 μg of BD™ Fc Block antibody (Cat. No. 553141 or 553142) for each 1 x 106 cells. Incubate on ice for 15 minutes.
- Aliquot an optimal concentration of fluorochrome-conjugated antibodies to each tube and add 100 μL (1 x 10 6) of cells.
- Incubate at room temperature for 30 minutes in the dark.
- Wash once with 2 mL of Stain Buffer and resuspend in the same buffer prior to flow cytometric analysis.
Protocol IV (Harsh Detergent Method)
Suggested Reagents
Full Name | Short Name | Catalog Number |
BD™ Phosflow Lyse/Fix Buffer I, 5x | Lyse/Fix Buffer I | 558049 |
BD™ Phosflow Perm Buffer IV, 10x | Perm Buffer IV | 560746 |
BD Pharmingen™ Stain Buffer (FBS) | Stain Buffer | 554656 |
Procedural Notes
- Be sure to remove all the supernatant after each centrifugation step. High residual volumes of supernatant will dilute the Perm Buffer IV, which could result in poor intracellular staining profiles.
- A longer than recommended permeabilization time, or using a ratio of cell to buffer volume outside the recommended ratio, could result in poorer fluorescent surface marker or phosphorylated protein specific antibody staining and detection.
- Permeabilization with Perm Buffer IV could result in decreased cell recovery, increase in time of permeabilization with Perm Buffer IV will increase cell loss.
- If staining of cell surface markers after permeabilization with Perm Buffer IV does not work well (there is a dim fluorescent signal), you can stain cells with fluorescent antibodies before cellular fixation (before step 4) or between the fixation and permeabilization steps (after step 9) see below.
Procedure
- Warm Lyse/Fix Buffer I to 37°C.
- Dilute Perm Buffer IV to 1.0x using 1x Phosphate Buffered Saline (PBS) prior to use.
- Prepare single-cell suspensions from mouse thymus or spleen in PBS with 5% FBS (no azide).
- Wash once with PBS with 5% FBS. Keep the cells at 4°C before stimulation.
- Treat the cells (example: use 100 μl of cell suspension) with the selected activator and/or inhibitor for the selected period of time.
- After treatment, add a 20-fold excess volume (eg, 2.0 mL) of warmed Lyse/Fix Buffer I. Mix well and incubate the tubes in a 37°C water bath for 10 to 15 minutes.
- Centrifuge the cells at 400g for 10 minutes in a tabletop centrifuge. Aspirate the supernatant.
- Wash the fixed cells once with PBS. Centrifuge at 400g and aspirate the supernatant.
- Vortex the tubes to loosen the cells.
- Permeabilize the cells by slowly adding Perm Buffer IV drop by drop to the cells. Add approximately 10 mL of Perm Buffer IV per 5 to 10 million cells. For cell concentrations less than 5 million, add a minimum of 1 mL of Perm Buffer IV.
- Incubate at room temperature for 15 to 20 minutes.
- Centrifuge at 400g for 10 minutes and aspirate the supernatant.
- Wash twice by adding Stain Buffer (FBS) to the cells. Centrifuge at 400g for 10 minutes and aspirate the supernatant each time.
- Resuspend the cells in Stain Buffer (FBS) at 10 million cells/mL and aliquot 100 μL to each sample tube.
- Aliquot an optimal concentration of phosphospecific, fluorochrome-conjugated antibody to each tube. Incubate at room temperature for 30 minutes in the dark.
- Wash once with 2 mL of Stain Buffer prior to flow cytometric analysis.
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.