This protocol compatibility table identifies the surface and intracellular antibodies that work with a common protocol. Find additional details about the content and navigating the table in the text at the bottom of the page.
Table Key: IC: Intracellular EC: Extracellular 
Recommended 
Decreased Performance 
Not Recommended 
Not Tested
Species Reactivity Key: H—Human M—Mouse R—Rat Mk—Monkey B—Bovine Pg—Pig Mi—Mink Dm—D. melanogaster Z—Zebrafish Sc—S. cerevisiae
| Protocol No. | Protocol Information | Brief Description of Staining Preparation Protocol | Available Fixation-Permeabilization Kits |
|---|---|---|---|
| 1 | Live Cell Surface Staining Protocol | Protocol where there is no fixation or permeabilization of the cells before staining | Not Applicable |
| 2 | Fixed Cell Intracellular Staining – Methanol Permeabilization Protocol | Uses formaldehyde and methanol for fixation and permeabilization of the cells before staining | Intracellular Flow Cytometry Kit (Methanol) #13593 |
| 3 | Fixed Cell Intracellular Staining – Triton X-100 Permeabilization Protocol | Uses formaldehyde and Triton X-100 for fixation and permeabilization of the cells prior to staining | Intracellular Flow Cytometry Kit (Triton X-100) #51995 |
| 4 | Fixed Cell Intracellular Staining – FoxP3/Transcription Factor Protocol | Fixation and permeabilization buffer set optimized for staining with antibodies to transcription factors and nuclear proteins | FoxP3/Transcription Factor Fixation/Permeabilization Kit #43481 |
There are many different protocols for you to choose from when staining either live or fixed cells in a flow cytometry experiment. Your protocols must be compatible with the antibodies used to stain the cells to avoid negatively affecting the epitope recognition while still allowing the antibody to access the cellular component where the protein of interest resides.
For example, staining intracellular targets for phospho-flow cytometry experiments requires that you fix and permeabilize your cells before staining. However, fixation (fix) and permeabilization (perm) protocols can alter the interaction between epitope and antibody. In particular, the choice of permeabilization reagent can have a significant influence on the sensitivity, specificity, and functionality of antibodies used within the assay. This can be both antigen- and clone-dependent.
Permeabilization is commonly achieved using solvents such as methanol or detergents like Triton X-100 or saponin. Saponin offers the least thorough permeabilization; it forms pores in the plasma membrane by associating with cholesterol but does not permeabilize intracellular membranes, such as the nuclear membrane or mitochondrial membrane. Triton X-100 and methanol will adequately permeabilize most intracellular membranes, with methanol providing the most thorough permeabilization. However, methanol can be disruptive to epitopes, and some antibodies, including some surface markers, will not work following methanol permeabilization.
Cell Signaling Technology® (CST) offers a diverse catalog of flow-cytometry validated antibodies conjugated with different fluorescent dyes. We’ve put together this Protocol Compatibility Table for Immune Signaling and Phenotyping by Flow Cytometry to help you quickly identify which staining protocols antibodies in the CST catalog are compatible with, especially when selecting multiple antibodies to use together in multiplex panels for signaling flow cytometry. Specifically, this table will help you understand:
Antibody Clone Information lists the CST antibody clone, epitope location, and species reactivity information for potential antibodies for your experiment.
Protocol Compatibility Testing identifies which of the most popular protocols were tested for each antibody clone to help you save time when building your panel. When selecting antibodies to multiplex in an experiment, you’ll want to make sure each clone is compatible with the same protocol. A green mark indicates the protocol is recommended, a yellow mark indicates the protocol was tested but may show decreased performance so further protocol optimization is recommended, a red mark indicates that the antibody is not recommended for that specific protocol, and a white mark means the protocol hasn’t been tested yet.
Available Conjugates lists the direct conjugate antibody products available from CST for each clone, organized from left to right by the approximate maximum emission of the respective fluorophore. Click on the linked product number to learn more about the specific antibody conjugate of interest.
