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Determination of Immune Cell Identity and Purity Using Epigenetic-Based Quantitative PCR
Published on: February 19, 2020
Quantitative single cell methods that identify cytokine and chemokine expression in dendritic cells
K Loré1, A L Spetz, T E Fehniger
1Department of Immunology, Microbiology, and Pathology, Division of Clinical Virology, Karolinska Institutet, Huddinge University Hospital, S-141 86 Huddinge, Stockholm, Sweden. karin.lore@medhs.ki.se
Journal of Immunological Methods
|February 28, 2001
Summary
Flow cytometry (FCM) and in situ imaging quantify dendritic cell (DC) cytokine expression. FCM is faster but less accurate for Golgi-localized proteins like IL-8, while in situ imaging is better for these specific markers.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- Dendritic cells (DCs) play a crucial role in immune responses.
- Accurate quantification of intracellular cytokine and chemokine expression in DCs is vital for understanding immune function.
- Existing methods for protein expression analysis have limitations.
Purpose of the Study:
- To develop and compare two techniques for quantifying intracellular cytokine and chemokine expression in DCs: flow cytometry (FCM) and in situ image analysis.
- To evaluate the qualitative and quantitative differences between FCM and in situ image analysis for detecting IL-8 and IL-1ra in DCs.
- To determine the optimal method based on protein localization and study objectives.
Main Methods:
- In vitro differentiated DCs were stimulated with lipopolysaccharide (LPS).
- Cells were stained for intracellular IL-8 (Golgi-localized) and IL-1ra (diffusely cytoplasmic).
- Protein expression was analyzed using both FCM and in situ image analysis, with and without Brefeldin-A treatment.
Main Results:
- DCs expressed IL-8 and IL-1ra with distinct staining patterns.
- FCM and in situ imaging showed similar frequencies for diffuse IL-1ra but varied for Golgi-localized IL-8.
- In situ imaging detected higher frequencies of IL-8 positive DCs compared to FCM.
- Brefeldin-A treatment improved detection for both methods, especially for IL-8, leading to similar frequencies.
Conclusions:
- FCM is faster and allows for multicolor analysis but is less accurate for cytokines with juxtanuclear staining patterns (e.g., IL-8).
- In situ image analysis is more accurate for quantifying proteins with specific localization patterns like IL-8.
- The choice between FCM and in situ image analysis depends on the specific cytokine, its localization, and the research question.
