IGC

Tips for Flow-ers

In this section we provide several tips for your flow cytometry experiments, from sample preparation to cell sorting conditions.

  • Designing experiments

    Use the Fluorescence viewers provided by BD Biosciences, Life Technologies, BioLegend, etc to select the right combination of fluorophores for the equipment you will be using in your flow cytometry experiment.

    You can find the SpetraViewers and other useful link in the Related Websites section.

  • How many cells should I acquire for analysis?

    Counting cells follows Poisson statistics:

    poisson

    where precision is defined by:

    cv

    As such to calculate the number of cells to acquire for analysis you should follow the example:

    example

  • Sorting experiments

    If you are thinking about sorting or already booked on a sorter please discuss the following issues with the operator:

    sortissues

    Improve sorting results

    Improve sample quality for better sorting results by avoid clump formation (ressuspend cells in PBS Ca/Mg++ free, 1mM EDTA, 25mM HEPES pH 7.0, 1-2% FBS) and by filtering ALL samples just before sorting (we provide cell strainers ith mesh size 30 μm).

    For Sticky Cells Raise concentration of EDTA to 5mM, use FBS dialyzed against Ca/Mg2+free PBS.

    For Adherent Cells use cation-free PBS/FBS buffer to inactivate trypsin (cations promote adherence of cells to plate and to each other). Level of EDTA can be increased if necessary but too much EDTA can be deleterious.

    Samples with high percentage of Dead Cells it is likely that there is soluble DNA from dead cells leading to severe clumping. Add 10 U/mL DNAse II to the buffer recipe.

  • Suggested Concentration Ranges

    *Based on “Guidelines for Cell Sorting”

    concentration

  • Sorting speed

    speedsort

  • How many cells do I need to sort (in purity mode)?

    totalcells

  • How long will my sorting take?

    durationsort