The Power of Reagent Titration in Flow Cytometry
Flow cytometry facilitates the detection of multiple cell parameters simultaneously with a high level of resolution and throughput, enabling in-depth immunological evaluations. High data resolution in flow cytometry depends on multiple factors, including the concentration of reagents used in the sta...
Saved in:
| Main Authors: | , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2024-10-01
|
| Series: | Cells |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2073-4409/13/20/1677 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1850205093726519296 |
|---|---|
| author | Diana L. Bonilla Alberta Paul Jesus Gil-Pulido Lily M. Park Maria C. Jaimes |
| author_facet | Diana L. Bonilla Alberta Paul Jesus Gil-Pulido Lily M. Park Maria C. Jaimes |
| author_sort | Diana L. Bonilla |
| collection | DOAJ |
| description | Flow cytometry facilitates the detection of multiple cell parameters simultaneously with a high level of resolution and throughput, enabling in-depth immunological evaluations. High data resolution in flow cytometry depends on multiple factors, including the concentration of reagents used in the staining protocol, and reagent validation and titration should be the first step in any assay optimization. Titration is the process of finding the concentration of the reagent that best resolves a positive signal from the background, with the saturation of all binding sites, and minimal antibody excess. The titration process involves the evaluation of serial reagent dilutions in cells expressing the antigen target for the tested antibody. The concentration of antibody that provides the highest signal to noise ratio is calculated by plotting the percentage of positive cells and the intensity of the fluorescence of the stained cells with respect to the negative events, in a concentration–response curve. The determination of the optimal antibody concentration is necessary to ensure reliable and reproducible results and is required for each sample type, reagent clone and lot, as well as the methods used for cell collection, staining, and storage conditions. If the antibody dilution is too low, the signal will be too weak to be accurately determined, leading to suboptimal data resolution, high variability across measurements, and the underestimation of the frequency of cells expressing a specific marker. The use of excess antibodies could lead to non-specific binding, reagent misuse, and detector overloading with the signal off scale and higher spillover spreading. In this publication, we summarized the titration fundamentals and best practices, and evaluated the impact of using a different instrument, sample, staining, acquisition, and analysis conditions in the selection of the optimal titer and population resolution. |
| format | Article |
| id | doaj-art-6faa8094494747dbbd05c87c9fbc9c6c |
| institution | OA Journals |
| issn | 2073-4409 |
| language | English |
| publishDate | 2024-10-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Cells |
| spelling | doaj-art-6faa8094494747dbbd05c87c9fbc9c6c2025-08-20T02:11:09ZengMDPI AGCells2073-44092024-10-011320167710.3390/cells13201677The Power of Reagent Titration in Flow CytometryDiana L. Bonilla0Alberta Paul1Jesus Gil-Pulido2Lily M. Park3Maria C. Jaimes4Scientific Commercialization, Cytek Biosciences, Inc., 47215 Lakeview Boulevard, Fremont, CA 94538, USAScientific Commercialization, Cytek Biosciences, Inc., 47215 Lakeview Boulevard, Fremont, CA 94538, USACustomer Education, Cytek Biosciences, Inc., 47215 Lakeview Boulevard, Fremont, CA 94538, USAScientific Commercialization, Cytek Biosciences, Inc., 47215 Lakeview Boulevard, Fremont, CA 94538, USAScientific Commercialization, Cytek Biosciences, Inc., 47215 Lakeview Boulevard, Fremont, CA 94538, USAFlow cytometry facilitates the detection of multiple cell parameters simultaneously with a high level of resolution and throughput, enabling in-depth immunological evaluations. High data resolution in flow cytometry depends on multiple factors, including the concentration of reagents used in the staining protocol, and reagent validation and titration should be the first step in any assay optimization. Titration is the process of finding the concentration of the reagent that best resolves a positive signal from the background, with the saturation of all binding sites, and minimal antibody excess. The titration process involves the evaluation of serial reagent dilutions in cells expressing the antigen target for the tested antibody. The concentration of antibody that provides the highest signal to noise ratio is calculated by plotting the percentage of positive cells and the intensity of the fluorescence of the stained cells with respect to the negative events, in a concentration–response curve. The determination of the optimal antibody concentration is necessary to ensure reliable and reproducible results and is required for each sample type, reagent clone and lot, as well as the methods used for cell collection, staining, and storage conditions. If the antibody dilution is too low, the signal will be too weak to be accurately determined, leading to suboptimal data resolution, high variability across measurements, and the underestimation of the frequency of cells expressing a specific marker. The use of excess antibodies could lead to non-specific binding, reagent misuse, and detector overloading with the signal off scale and higher spillover spreading. In this publication, we summarized the titration fundamentals and best practices, and evaluated the impact of using a different instrument, sample, staining, acquisition, and analysis conditions in the selection of the optimal titer and population resolution.https://www.mdpi.com/2073-4409/13/20/1677flow cytometryantibody titrationspectral cytometrydata reproducibility |
| spellingShingle | Diana L. Bonilla Alberta Paul Jesus Gil-Pulido Lily M. Park Maria C. Jaimes The Power of Reagent Titration in Flow Cytometry Cells flow cytometry antibody titration spectral cytometry data reproducibility |
| title | The Power of Reagent Titration in Flow Cytometry |
| title_full | The Power of Reagent Titration in Flow Cytometry |
| title_fullStr | The Power of Reagent Titration in Flow Cytometry |
| title_full_unstemmed | The Power of Reagent Titration in Flow Cytometry |
| title_short | The Power of Reagent Titration in Flow Cytometry |
| title_sort | power of reagent titration in flow cytometry |
| topic | flow cytometry antibody titration spectral cytometry data reproducibility |
| url | https://www.mdpi.com/2073-4409/13/20/1677 |
| work_keys_str_mv | AT dianalbonilla thepowerofreagenttitrationinflowcytometry AT albertapaul thepowerofreagenttitrationinflowcytometry AT jesusgilpulido thepowerofreagenttitrationinflowcytometry AT lilympark thepowerofreagenttitrationinflowcytometry AT mariacjaimes thepowerofreagenttitrationinflowcytometry AT dianalbonilla powerofreagenttitrationinflowcytometry AT albertapaul powerofreagenttitrationinflowcytometry AT jesusgilpulido powerofreagenttitrationinflowcytometry AT lilympark powerofreagenttitrationinflowcytometry AT mariacjaimes powerofreagenttitrationinflowcytometry |