Mouse Blood Immunophenotyping Panels for Low-Volume Flow Cytometry
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Solution Overview
Problem
Flow cytometry in mice faces limitations due to the limited availability of mouse antibodies, the need for small blood volumes, and the variability of immune cell populations, which complicates longitudinal studies and reduces the ability to analyze immune cell subpopulations effectively.
Innovation Solution
A novel method and reagent composition using a panel of fluorescently labeled antibodies, including markers such as CD45, Ly6C, B220, CCR2, CD11b, CD11c, CD172a, CD49b, CD80, CD86, F4/80, Ly6G, MHC-II, Siglec-F, or CD45, CCR4, CCR6, CD138, CD19, CD25, CD3, CD4, CD44, CD62L, CD8, CXCR3, TNF-RII, NK1.1, allowing analysis of immune cell populations from as little as 50-100 µl of peripheral blood, enabling detection of over 30 subpopulations and obtaining absolute cell counts.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If flow cytometry is performed on mouse peripheral blood to analyze immune cell populations, then comprehensive immunophenotyping data can be obtained, but the blood volume required exceeds 10% of the mouse's total blood volume
Solution Approach 1:
The blood sample is segmented into multiple aliquots, each stained with a specific panel of fluorescently labeled antibodies targeting different immune cell markers. This allows comprehensive analysis of multiple immune cell populations from a small total blood volume by dividing the sampling burden across multiple targeted assays rather than requiring one large-volume unstained sample
Solution Approach 2:
The invention changes the parameters of the flow cytometry assay by using highly sensitive fluorescently labeled antibodies and optimized staining protocols that maximize signal detection from minimal cell numbers. This enables accurate immunophenotyping with as little as 10% of the mouse's blood volume by enhancing the detection efficiency per unit volume
2Device complexity
If simple flow cytometry panels are used to reduce antibody requirements, then fewer markers can be detected, but the ability to identify diverse immune cell subpopulations is limited
Solution Approach 1:
The comprehensive immune phenotyping panel is segmented into multiple specialized antibody panels, each targeting specific immune cell lineages (e.g., T cells, B cells, myeloid cells, NK cells). Each panel contains fluorochrome-conjugated antibodies against specific markers for that cell type, allowing detailed analysis of individual populations while collectively covering the entire immune system across multiple assays
Solution Approach 2:
The invention creates a universal multi-panel system where fluorochrome-conjugated antibodies against various immune cell markers can be combined in different configurations. The same fluorescent dyes and basic assay protocol are used across multiple panels, providing versatility in detecting diverse immune cell populations while maintaining a standardized, manageable complexity through reusable components
3Measurement precision
If minority immune cell populations are analyzed in small blood volumes, then detection sensitivity is reduced, but variability increases due to external factors
Solution Approach 1:
The invention optimizes multiple parameters simultaneously: uses highly sensitive fluorochromes with high quantum yield, employs optimized antibody concentrations and incubation times, and implements standardized processing protocols. These parameter changes collectively enhance detection sensitivity for minority populations while reducing variability by minimizing the impact of external factors through controlled, reproducible assay conditions
4Duration of action of stationary object
If longitudinal studies are conducted with repeated blood extractions, then disease progression can be monitored, but the total blood volume extracted may harm the mouse
Solution Approach 1:
The longitudinal monitoring protocol is segmented into multiple time points with small blood volume extractions (≤10% of total blood volume) rather than requiring large single-time-point samples. Each extraction uses targeted antibody panels that maximize information gain from minimal cell numbers, enabling long-term disease progression monitoring while cumulative blood loss remains below harmful thresholds
Solution Approach 2:
The invention implements preliminary optimization of the assay protocol to minimize blood volume requirements before conducting longitudinal studies. By establishing highly efficient staining and detection procedures in advance, the study design ensures that subsequent repeated extractions can be performed at safe volume thresholds while still achieving comprehensive immunophenotyping data
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The method allows for comprehensive analysis of immune cell subpopulations in mice, facilitating longitudinal studies without stress to the animal and providing accurate, absolute values, overcoming previous limitations in volume and variability, and validating changes in immune populations using an LPS lung inflammation model.
Implementation Method 1
Flow cytometry (FCM) is a fluorescence-based technique that allows studying the optical properties of cells as well as quantifying them when they are suspended in a flow
Implementation Method 2
incubating the selected panel of fluorescently labeled antibodies with the blood sample to be tested
Data Source
Figure 1~1L
Figure 1M~1Q
AI summary
The present invention relates to a novel method for flow cytometric immunophenotyping of a mouse from a very small volume of blood using a novel combination of markers. The invention furthermore relates to a reagent composition for flow cytometric immunophenotyping of blood cells comprising fluorochrome-conjugated antibodies directed against a combination of markers.