1- Please describe a 5-color flow cytometry panel (specific colors) with a death dye that requires NO/or minimum compensation (The flow machine is the LSR for BD).
2- Please describe a flow cytometry typical gating strategy to quantify the protein levels of a protein conjugated with a fluorochrome (for example PE). List what software you typically use.
3- Please elaborate how you typically do immunofluorescence analysis (level of complexity and depth of analysis). This can be very time consuming and complex.
Saman Behboodi Tanourlouee
5-color flow cytometry panel with minimal compensation:Blue 488nm laser: FITC (Green) Yellow-green 561nm laser: PE (Orange) Red 640nm laser: APC (Far Red) Violet 405nm laser: Pacific Blue (Blue) Violet 405nm laser: Live/Dead stain like the amCyan or Aqua (specific colors often for dead cells) The idea here is to choose fluorochromes that have distinct emission spectra to reduce spectral overlap, hence reducing the need for compensation. Gating strategy to quantify protein levels conjugated with PE:Step 1: Forward Scatter (FSC) vs. Side Scatter (SSC) to gate on overall cells and exclude debris. Step 2: Apply a gate on Live/Dead staining (if used) to exclude dead cells. Step 3: FSC-Height vs. FSC-Area to exclude cell aggregates or doublets. Step 4: PE histogram or PE vs. another parameter (if dual staining). This will allow you to identify the intensity of PE signal which correlates with protein levels. Software: FlowJo is a common choice for analyzing flow cytometry data. Immunofluorescence analysis:Preparation: Start with fixation, often with paraformaldehyde, and then permeabilize cells (commonly with Triton X-100). Block non-specific binding sites (usually with a serum). Staining: Incubate your cells with primary antibody against your target protein, followed by a fluorochrome-conjugated secondary antibody. DAPI can be used to stain nuclei. Imaging: Using a fluorescence microscope, capture images ensuring you don't have any bleed-through between channels. Analysis: Software such as ImageJ (free) or FIJI can be used. Measure fluorescence intensity, co-localization, or count positive cells. Complexity: The depth of the analysis depends on the question at hand. If you're quantifying fluorescence intensity, make sure to subtract the background. If analyzing co-localization, consider using plugins like JACoP in ImageJ. Remember, while the basics are covered here, the devil's in the details. Always optimize and validate your experimental setup!
Hello Malihe Naderi ! Sure thing, let's get straight to it:
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