Flow cytometry detects physical and chemical properties of cells and is used in a broad range of applications including analysis of cell surface and intracellular markers. It requires protein marker-specific reagents such as primary antibodies conjugated to fluorescent dyes that label target proteins both outside (cell surface) and inside (intracellular) cells.
Detection of intracellular proteins by FC
Intracellular flow cytometry is primarily used for analysis of signalling and functional responses within cells including detection of cytokines (inflammation signalling and growth promoting proteins) and transcription factors (proteins that control gene activity) and phosphorylated signal transducers.
Detection of intracellular proteins depends on their biology and location in the cell and, for analysis using intracellular flow cytometry, a fixing and permeabilising protocol is required. Cells are firstly fixed or frozen in place using chemicals such as 4% paraformaldehyde or formaldehyde. This step stabilizes the cell membrane and preserves the overall cell structure. Cells are then permeabilised with a detergent or alcohol to allow fluorescently labelled conjugated antibodies to access and label the target intracellular proteins. Once fixed, permeabilised and labelled, the cells are then analysed by flow cytometry.
Reagents for intracellular flow cytometry
For intracellular flow cytometry, all protocols require the following reagents:
- Fluorescently labelled primary antibodies to the target protein
- Fix and perm(eabilisation) reagents
- Isotype controls – negative controls that measure background fluorescence caused by non-specific antibody binding. An isotype control is a primary antibody of the same isotype and host species, labelled with the same fluorophore as that used for target protein detection. However, the specificity of the antibody is not to the target protein
Proteintech's FC portfolio
Proteintech offers users a complete portfolio of easy-to-use products and protocols for intracellular flow cytometry single cell analysis.
Proteintech’s large range of primary antibodies target both cytoplasmic and nuclear proteins and are conjugated to either CoraLite®or APC fluorescent dye labels.
Proteintech’s optimized fixation and permeabilisation reagents ensures results are highly reproducible.
Their isotype controls enable accurate detection of target protein populations.
Intracellular flow cytometry products
Cytoplasmic and nuclear protein primary antibody conjugates: a wide range of poly- and monoclonal antibody conjugates are available labelled with different coloured CoraLites®or APC fluorescent dyes.
| Cat-No. | Product | Fluorophore | Host / Isotype |
|---|---|---|---|
| CL488-60004 | CoraLite®488-conjugated GAPDH Monoclonal antibody | CoraLite®488 | Mouse / IgG2b |
| CL488-65089 | CoraLite®488 Anti-Mouse Foxp3 (3G3) | CoraLite®488 | Mouse / IgG1 |
| CL488-66031 | CoraLite®488-conjugated Alpha Tubulin Monoclonal antibody | CoraLite®488 | Mouse / IgG2b |
| FITC-10835 | FITC-conjugated ATF4 Polyclonal antibody | FITC | Rabbit / IgG |
| CL488-65053 | CoraLite®488 Anti-Human CD107b (H4B4) | CoraLite®488 | Mouse / IgG1 |
| CL488-66251 | CoraLite®488-conjugated 14-3-3 Sigma Monoclonal antibody | CoraLite®488 | Mouse / IgG1 |
| CL488-66020 | CoraLite®488-conjugated Bcl-XL Monoclonal antibody | CoraLite®488 | Mouse / IgG2b |
Isotype controls: available in unconjugated, APC or CoraLite fluorescent dye formats.
| Art-No. | Isotype control | Fluorophore | Species / Isotype |
|---|---|---|---|
| 30000-0-AP | Rabbit IgG control Polyclonal antibody | Unconjugated | Rabbit / IgG |
| 66360-1-Ig | Mouse IgG1 isotype control Monoclonal antibody | Unconjugated | Mouse / IgG1 |
| 66360-3-Ig | Mouse IgG2b isotype control Monoclonal antibody | Unconjugated | Mouse / IgG2b |
| CL647-65212 | CoraLite®647 Rat IgG1 Isotype Control (HRPN) | CoraLite®647 | Rat / IgG1, κ |
| CL647-65209 | CoraLite®647 Rat IgG2a Isotype Control (2A3) | CoraLite®647 | Rat / IgG2a |
Fix and Perm Reagents: formulated buffers and solutions that are easy to use and provide consistent results.
| Art-No. | Item | Description |
|---|---|---|
| PF00011 | Foxp3 / Transcription Factor Staining Buffer Kit | Buffers and solutions for detection of nuclear antigens such as Foxp3 and ROR gamma |
| PF00011-A | Foxp3 / Transcription Factor Fix-Perm Concentrate (4X) | Concentrated solution for dilution with Foxp3 / Transcription Factor Fix/Perm Diluent |
| PF00011-C | Flow Cytometry Perm Buffer (10X) | For use for intracellular staining of cytokines and other cytoplasmic antigens |
| PF00012 | Flow Cytometry Staining Buffer (1X) | For use for diluting antibody and cell suspensions and as a wash buffer |
| PF00013 | RBC Lysis Buffer (10X) | For lysing red blood cells in single cell suspensions |