研究目的
To develop a method based on imaging flow cytometry for localizing miR-590-5p with fluorescent probes in dendritic cells (DCs) to study miRNA processing in DCs.
研究成果
The method based on imaging flow cytometry provides an additional method to study miRNA processing in DCs, which makes it a valuable addition to existing miRNA research techniques.
研究不足
The limitation of this method is that cells in suspension are required because of the flow cytometry technology. Evaluating endocytosis or RNA interference in primary tissues therefore remains a challenge.
1：Experimental Design and Method Selection:
A method based on imaging flow cytometry was developed to localize miR-590-5p with fluorescent probes in DCs.
2：Sample Selection and Data Sources:
Mouse bone marrow DCs (BMDCs) were isolated from the femur bone marrow of C57/BL6J mice.
3：List of Experimental Equipment and Materials:
ImageStreamX MkII (Merck Millipore, Seattle, WA) one camera system with six lasers was used for the imaging flow cytometry assay.
4：Experimental Procedures and Operational Workflow:
DCs were incubated with miR-590-5p-Cy3 and miR-mock-Fam-F for different durations, and the internalization ratios were measured.
5：Data Analysis Methods:
Analysis was performed on the compensated image files using algorithms in IDEAS image analysis software.
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ImageStreamX MkII
MkII
Merck Millipore
Used for the imaging flow cytometry assay to quantify, visualize and localize the fluorescence intensity in individual cells.
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CD11c magnetic beads
Miltenyi
Used for sorting CD11c+ cells from bone marrow cells.
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RPMI1640 media
Used for culturing bone marrow cells and DCs.
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Fetal Bovine Serum
Used as a supplement in RPMI1640 media for culturing cells.
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GM-CSF
R&D Systems
Used in the culture medium for bone marrow cells to promote DC differentiation.
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IL-4
R&D Systems
Used in the culture medium for bone marrow cells to promote DC differentiation.
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LPS
Sigma-Aldrich
Used to activate DCs.
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TRIzol
Life Technologies
Used for total RNA extraction from DCs.
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Reverse Transcript Kit
QIAGEN
Used for reverse transcription of RNA.
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miScript SYBR Green qPCR kit
QIAGEN
Used for qPCR to measure miRNA levels.
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Lipofectamine 2000
Invitrogen
Used for transfection of miRNA mimics into cells.
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BCA Protein Assay Kit
Pierce
Used for protein quantification.
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SDS-PAGE
Used for protein separation.
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Polyvinylidene difluoride membranes
Used for protein transfer in western blotting.
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ECL reagent
Thermo
Used for developing western blots.
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FC2 Image Station
Alpha
Used for capturing images of western blots.
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