Abstract: Determination of suitable antibody concentration is an important problem in immunophenotype. Excessive or insufficient staining reagents can cause increased non-specific staining and experimental cost, and decreased signal noise ratio. The titration process effectively analyzes the positive background signal with the minimum antibody content saturating all binding sites. The evaluation of antibody performance in the target cell follows the recommended dilution ratio below: 1X, 2X(6-12 serial 2-fold dilutions).
Keywords: Antibody Titration, Flow Cytometry, Serial Titration, Combinatorial Titration
1. Signal Noise Ratio Calculation
Various methods can evaluate and determine the titer. The simplest method is signal noise ratio(SNR) calculation, which is the mean fluorescence intensity(MFI) ratio between positive and negative group. The best titer is the highest SNR and can effectively distinguish positive and negative signal. This method can accurately determine the best antibody concentration and improve the experimental sensitivity and reliability.
2. Staining Index(SI) Antibody Titration
This method first calculates the MFI coefficient between positive and negative group. Then, divide the trebled standard deviation by MFI coefficient. The best titer is the highest SI value, optimizing the experimental accuracy and reliability.
3. Serial Titration
Dead cells in multi-parameter and color flow cytometry can interfere with results. Insufficient positive cells in complex samples make evaluation of the best titer difficult. Serial titration is very suitable for multicolor labeled samples with complex cell types and many dead cells. This method first confirms the best concentration of reactive dyes and lineage markers. Then, titrate other markers to obtain the best titer.
4. Combinatorial Titration
Since applications of multi-parameter and color flow cytometry are very wide, workload of single titration for each antibody is huge. Combinatorial antibody titration can simply procedure to save a lot of time and costs without affecting data quality. The efficiency of establishing flow cytometry is obvious improved.
Notes:
● During antibody titration, the sample should contain a certain proportion of negative cells to accurately evaluate background staining. For lower proportion of negative cells, add unstained cells to observe background fluorescence.
● Besides, sufficient positive cells can accurately evaluate the best antibody concentration. For fewer positive cells, conduct serial titration in specific subgroup.
● The best titer should be shown in concentration of stained antibody instead of dilution ratio.
● Changes in staining method, antibody lot or storage condition require for new titration.
● The best concentration from single titration may be adjusted for staining, antibody competition and multicolor resolution loss.
● During combinatorial titration, assign fluorescent-dye conjugated antibodies with less overlapped spectrum to the same group.
● During serial or combinatorial titration, antibody volume can dilute other antibodies. Thus, higher antibody concentration can reduce dilution effects.
| Recommended Products | |||
| Species | Cell Populations | Flow Cytometry Antibody Combination | Cat.No |
| Human | T/B/NK cell populations detection | CD45-PerCP | PCP-30039 |
| CD3-FITC | FITC-30004 | ||
| CD16-PE | PE-30061 | ||
| CD56-PE | PE-30008 | ||
| CD19-APC | APC-30066 | ||
| Human | Thl/Th2 cell populations detection | CD3-PerCP/Cyanine5.5 | PCP55-30004 |
| CD4-FITC | FITC-30005 | ||
| IFN-γ-PE | PE-30053 | ||
| IL4-APC | APC-30043 | ||
| Mouse | Thl/Th2 cell populations detection | CD3-PerCP/Cyanine5.5 | PCP55-30002 |
| CD4-FITC | FITC-30128 | ||
| IFN-γ-PE | PE-30074 | ||
| IL4-APC | APC-30026 | ||
| Human | Treg cell populations detection | CD4-FITC | FITC-30005 |
| CD25-PE | PE-30035 | ||
| CD3-PerCP-Cy5.5 | PCP55-30004 | ||
| CD127-FineTest®647 | F647-30033 | ||
| Mouse | Treg cell populations detection | CD4-FITC | FITC-30128 |
| CD25-APC | APC-30017 | ||
| FOXP3-PE | PE-30111 | ||
REFERENCES
[1]Panel Optimization for High-Dimensional Immunophenotyping Assays Using Full-Spectrum Flow Cytometry, PMID: 34492732.
[2]Combinatorial antibody titrations for high-parameter flow cytometry, PMID: 38317641.