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Performing basic scRNA-seq​1. Separating live single cells from tissues of interest2. Capture as much RNA as possible by lysing isolated cells - usually using poly[T]-primer3. Transform poly[T]-primed RNA into cDNA with reverse transcriptase (adding adapter sequences for sequencing platforms used and barcoding tasks for cell origin - adding unique molecular identifieres (UMIs))4. cDNA amplificat..

1. Immune Response and Immunophenotyping Objective: Understanding immune cell dynamics and responses, particularly in infection and inflammation. Application: Flow cytometry is used to identify different immune cell populations and analyze their surface markers. It helps track the behavior of T cells, B cells, and monocytes in various conditions. Example Article: "Flow cytometry-based identifica..

There are a wide variety of experiments that can be performed using flow cytometry.1) Experiment using fluorescent antibodiesAs we have already covered to some extent when introducing flow cytometry, the main purpose of flow cytometry is to find out and analyze what kind of cell it is by analyzing various proteins expressed by the cell. Therefore, many fluorescent antibodies that can specificall..

Principle of FluorescenceFluorescent dyes have wavelengths of light absorbed by each dye.If you shine light in the corresponding wavelength range of light, you can excite electrons in fluorescent dye.Excited electrons go to a higher energy state, an excited state, and then go back to a lower energy state, a ground state.At this time, the energy emitted is emitted in the form of photons.Through t..

Sample PreparationTissue fixation and permeabilization are the two most important aspects of sample preparation for FISH, significantly impacting the quality of the data extracted. Fixation is commonly achieved using 4% paraformaldehyde (PFA), which should be prepared fresh for each usage as PFA in solution forms polymers with long-term storage. An alternative is to use a 1:10 dilution of formal..