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Flow cytometric determination of CD11b upregulation in vivo
H Repo1, S E Jansson, M Leirisalo-Repo
1Department of Bacteriology and Immunology, University of Helsinki, Finland.
Insights
This study presents a new flow cytometry method to measure CD11b integrin upregulation on neutrophils and monocytes. The optimized technique ensures cell viability and accurately reflects in vivo CD11b expression levels in patients.
Area of Science:
- Immunology
- Cell Biology
- Flow Cytometry
Background:
- Integrin CD11b plays a crucial role in neutrophil and monocyte function.
- Accurate measurement of CD11b upregulation in vivo is essential for understanding inflammatory responses.
- Previous methods were susceptible to in vitro artifacts affecting CD11b expression.
Purpose of the Study:
- To develop and validate a flow cytometric method for evaluating in vivo CD11b integrin upregulation on peripheral blood neutrophils and monocytes.
- To establish optimal conditions for cell handling to prevent spontaneous in vitro CD11b upregulation.
- To assess CD11b expression levels in specific patient populations.
Main Methods:
- Developed a low-temperature (0-4°C) flow cytometry protocol using specific monoclonal antibodies (CD11b PE, CD14 FITC) and a nucleic acid dye (LDS-751).
- Employed live-cell gating strategies based on LDS-751 staining and light scattering to exclude non-viable and aggregated cells.
- Investigated the impact of temperature, pre-fixation, and density gradient separation on CD11b expression.
Main Results:
- The optimized method maintained high leukocyte viability (95-100%).
- Maximal CD11b expression was observed on neutrophils and CD14+ cells from arthritic synovial fluid.
- Increased, submaximal CD11b levels were detected on peripheral blood neutrophils and monocytes from sepsis recovery patients.
Conclusions:
- The described flow cytometry method reliably quantifies in vivo CD11b upregulation on neutrophils and monocytes.
- The protocol minimizes in vitro artifacts, ensuring accurate assessment of CD11b expression.
- This method is valuable for studying inflammatory conditions and immune cell activation in clinical settings.
Abstract:
We describe a flow cytometric method to evaluate upregulation of peripheral blood neutrophil and monocyte integrin CD11b in vivo. To avoid spontaneous upregulation in vitro, buffy coat cells were separated on ice and all subsequent cell handling steps were carried out at 0-4 degrees C. Such leukocytes were 95-100% viable, as determined by PI staining. Buffy coat leukocytes were double-stained with CD11b PE-conjugated and CD14 FITC-conjugated monoclonal antibodies and, in addition, with the nucleic acid dye LDS-751. After staining, firstly, LDS-751 positive (+ve) leukocytes, and, secondly, CD14 +ve monocytes were collected in live mode. Aggregated and irrelevant cells were gated out on the basis of their LDS-751 staining pattern and cellular light scattering properties, and the CD11b expression on neutrophils and monocytes was determined. Upregulation of CD11b in vitro was significantly affected by factors such as cell handling temperature, pre-fixation of blood samples, and density gradient separation of the cells. Incubation of aliquots of buffy coat cell suspension supplemented with FMLP for 5 min or without FMLP supplement for 15 min at 37 degrees C significantly increased CD11b expression without affecting cell viability. We have demonstrated that CD11b is expressed at maximal levels on arthritic synovial fluid neutrophils and CD14 +ve cells, and at increased but submaximal levels on peripheral blood neutrophils and monocytes of patients recovering from sepsis. The results suggest that the method can be used to evaluate in vivo upregulation of CD11b.