Flow Post-Its

Looking for tips and tricks on how to improve your flow cytometry experiments? Maybe you're unsure about certain aspects of the technology and need more information? Check out our Flow Post-it program! Born at Memorial Sloan Kettering Cancer Center, it is now a collaborative educational program with the Whitehead Institute for Biomedical Research.

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Post-Its
Flow Post-it Threshold

An explanation of what threshold is in flow cytometry and how adjustments can impact your data.

Flow Post-it Nuclei Sorting

Looking to sort out nuclei? This flow post-it is for you!

Under Pressure Flow Postit

Description of how sample pressure and sheath pressure work on flow cytometers. 

Histogram or Dot Plots

06/21/21

An explanation in the differences between histograms and dot plots and population identification.

Flow Post-IT HRP Validation
06/01/21

Method to validate deposition of droplets when using flow cytometry to purify cells into plates on a cell sorter.

Flow Post-it Pulse Processing
05/10/21

How to utilize the pulse processing to gate out aggregates of cells and ensure more robust analysis?

Flow Post-it Optimizing Voltage_Gain
04/19/21

Setting PMT voltages or APD gains appropriately is a critical step for a successful flow cytometry experiment

Flow Post-It JPEG
03/29/21

Exposure of cells to laser light in flow cytometers causes molecules such as NADPH & flavins to emit fluorescence. This intrinsic emission of light is what we call autofluorescence (AF). Particles or cells can have varying levels of autofluorescence, and each cell or condition type should have a fully unstained control to account for this.

Flow Post-it Cell Dissocation
03/08/21

Cell Dissociation: One Size Does NOT Fit All

Flow Post-it Single Stain Controls and Gating
02/15/21

On the importance of proper gating negative and positive populations in single stain controls for compensation or unmxing

Flow Post-It JPEG
01/25/21

When analyzing multiparameter flow cytometry data where spillover correction has been carried out, a quality check should always be done to determine the accuracy of results. Visualizing NxN plots with the appropriate scaling to view all events is necessary when doing these quality assessments. Extreme negatives are typically indicative of overcompensation or overestimation of unmixing.

Flow Post-It JPEG
12/20/20

DAPI (4′,6-diamidino-2-phenylindole) is a fluorescent stain often used to differentiate between live and dead cells for viability measurements in flow cytometry. This reagent is a popular choice due to its short incubation time and high relative brightness. Similar to other reagents, DAPI staining conditions must be optimized for best experimental results.

Flow Post-it Washes
12/07/20

How the amount of washing steps influence your staining?

Flow Post-It JPEG
11/16/20

When designing and executing multiparameter flow cytometry experiments, instrument configuration and panel design both play a very important role. Just because a cytometer can detect fluorochromes does not mean that it will work with your experiment. Care must be taken when designing your panel to avoid fluorochrome combinations that introduce high spread with coexpressing markers.

Flow Post-it Apoptosis
11/03/20

How to perform the study of the cell changes during apoptosis with flow cytometry?

Flow Post-It JPEG
10/19/20

In order to accurately analyze multicolor flow cytometry data, spectral overlap must be corrected with compensation or unmixing. When running the single stain controls, it is essential that the fluorochromes match those in your experimental samples. Failure to follow this best practice will result in incorrect data.

Flow Post-it Minimizing Aggregates

10/5/20

Why are Single Cell Suspensions Important? 

Scaling Flow Post-it

09/21/20

Comparison of scaling across same sample

Index Sorting

09/08/20

What is Index sorting and when can I use it?

FMO

08/25/20

Why FMOs are so essential to set appropriate gates?

Recycling Compensation

08/10/20 

Should old compensation matrices be reused from experiment to experiment?

 

Flow Post-It JPEG
07/27/2020

In multicolor Flow Cytometry experiments, Spillover Spread translates into the width of the positive population in adjacent detectors, and is one of the most important factors impacting resolution. The amount of spillover spread is commonly misunderstood as being a result of compensation or unmixing, but this is not the case. Spreading is a result of photon counting error, prior to any processing of the data such as compensation. The spillover value, on the other hand, is a result of amount of overlap and detector gains, among others. Therefore, independent of spread and has no impact on resolution.

Flow Post-It JPEG
07/13/2020

In order to be confident in your sorting results, cell sorter performance must be evaluated through the appropriate QC methods. Rmax is a method that calculates the maximum recovery of the sort sample by looking at how much is lost in the unsorted fraction. Deviations from maximum recovery indicate a problem in sorter performance.

Flow Post-It JPEG
06/29/2020

Flow Cytometry commonly utilizes antibodies conjugated to fluorochromes as a means of identifying subsets of cells within a heterogenous sample. Purchasing from a vendor does not guarantee the functionality of the antibody. Validation must be carried out for all antibodies to allow rigor and reproducibility in your Flow Cytometry experiments.

Flow Post-It JPEG
06/15/20

When conjugating two dyes where one fluorochrome’s emission spectra (donor) overlaps the excitation spectra of a second fluorochrome (acceptor) a phenomenon called Förster Resonance Energy Transfer (FRET) occurs, creating a new dye with the excitation maximum of the donor and the emission maxima of the acceptor. The resulting dye is called a Tandem dye.

Flow Post-It JPEG
06/01/2020

When carrying out compensation or unmixing in multicolor Flow Cytometry experiments, the use of the appropriate controls is critical. Both beads and cells are often used in these corrections for fluorescence spillover, often times in combination. It is necessary in these cases to have the appropriate autofluorescence referenced for each single color for acquisition and analysis softwares to accurately correct for spillover.

Flow Post-It JPEG
05/18/2020

Studying Cell cycle by Flow Cytometry can be performed by staining cells or nuclei with fluorescent DNA markers or nucleoside analogs and measuring the signal output. By using DNA dyes that bind stoichiometrically we can assess DNA ploidy level, cell cycle
stage, the presence of apoptotic cells and performance of drugs for treatment of disease states.

Flow Post-It JPEG
05/04/2020

Compensation and unmixing are common methods used to determine the true amount of fluorescence from a fluorochrome when using either traditional or full spectrum Flow Cytometry to carry out multicolor experiments. In these cases, it is necessary to use appro priate single color controls which adhere to all the appropriate rules. If any of these rules are not met compensation or unmixing may not be cor rect. To troubleshoot the acquired panel we need to use a quantitative approach to determine if compensation and unmixing are correctly determined.

Flow Post-It JPEG
04/20/2020

 Antibody Titration is one of the most important steps in Panel optimization to ensure best Resolution. It allows you to find the optimal concentration of antibody that results in the brightest signal of the positive population while avoiding background staining, thus maximizing signal-to-noise ratio. Additionally, it helps save money and reagents.

Flow Post-It JPEG
04/02/2020

Why should we use vital dyes? Distinguishing debris from small cells in tissue preps for Flow Cytometry can often be difficult. Dead cell removal (through nuclear or amine reactive viability dyes) and scatter gating alone cannot be used in scenarios such as these to pull out live cells for analysis or sorting.

Flow Post-It JPEG
03/23/20

In Cancer Research, assessing Cell Proliferation can be useful for a variety of applications, such as cytotoxicity assays, CAR-T cell expansion, drug development, and determining inhibition of tumor growth.

Fixable Viability Dye

3/9/20

Details on fixable viability dyes for Flow Cytometry and how they can be used in your experiments

Flow Post-It JPEG
02/22/20

An antibody or immunoglobulin is a Y-shaped protein produced by plasma cells (Fig.1) that serve a variety of immunological functions. The antibody recognizes a specific structure called an antigen (i.e cell surface marker or pathogen), via the unique fragment antigen-binding (Fab) region. The fragment crystallizable (Fc) region can interact with cell surface receptors (Fc receptors) for a variety of biological functions. Fc receptors can be found on subsets such as: B lymphocytes, dendritic cells, monocytes, macrophages, NKs, neutrophils, eosinophils, human platelets, mast cells and basophils.

Flow Post-It JPEG
02/10/20

Cell Viability, autofluorescence and cell aggregation may all affect the quality of cell sorting experiments. Good sample preparation is crucial and will result in better sort purity, yield and post-sort cell function and viability.

Cell Proliferation by Flow Cytometry

Flow Post-It JPEG
01/20/20

Flow Cytometry measures the properties of cells and particles in a stream of fluid, allowing multiparametric analysis at a single-cell level. Fluorescently- labeled cells in suspension are run on flow cytometers where they pass in file, one by one, through one or more lasers of different wavelengths. Scattered laser light or emitted fluorescence are collected and transmitted through optical pathways and amplified/digitized for downstream analysis.