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Show tips for selecting fluorochromes for multicolor flow cytometry

Research the fluorochrome properties - ex. tandem dyes are sensitive to photobleaching and can become uncoupled; some fluorochromes are better suited for intracellular staining than others (ex. PE-Cy™5 can withstand the harsh washing conditions required for intracellular staining).

To limit the amount of compensation required, select a single fluorochrome in each laser excitation range, rather than exciting multiple fluorochromes with the same laser.

Choose fluorochromes with the least amount of spectral overlap.

Select the brightest fluorochromes that can be detected by your instrument.

Pair the brightest fluorochromes with the lower expressing proteins and the dimmer fluorochromes with the highly expressed proteins.

Launch the Flow Animation Video

Traditionally, flow cytometry has been used to identify distinct cell types within a heterogeneous pool of cells, based on extracellular or surface marker expression, an application commonly known as immuno-phenotyping. However, this technology is also readily amenable to intracellular target detection and can be successfully applied to the study of complex signaling events.

Thus combined with the detection of cell surface markers intracellular flow cytometry has evolved into a powerful platform, which enables characterization of signaling networks at a single cell level within phenotypically distinct cell populations.

Advantages of Intracellular Flow Cytometry:

  • Multi-parameter (e.g., multiple intra- and extra-cellular targets, viability, DNA content, cytokine profile, etc.)
  • Sensitivity (single cell analysis)
  • Quantitative output
  • High throughput (up to a 1000 cells/sec can be processed)
  • Fast readout (min to hours to generate data)

Intracellular Flow versus Western blot data (p-Syk)

Intracellular flow cytometry offers unique advantages to the study of signaling events when compared to western blot analysis. Western blots detect protein levels from a pooled population of cells and therefore may not be sufficiently sensitive to recognize infrequent signaling events, particularly those taking place in a small proportion of specialized cells. This can be misconstrued as a negative result. Owing to its capacity to measure events at a single cell level, flow cytometry allows for the identification of rare signaling events (see example below).

Western Blot

Western Blot

Intracellular Flow Cytometry

Syk, a non-receptor cytoplasmic tyrosine kinase transmits signals from the B cell receptor (BCR) and is phosphorylated upon BCR activation. WB was used to study activation of Syk in a PBMC population, which was left either untreated or was treated with IgM (10 μg/ml) for 10 min. Analysis of total Syk by WB reveals two bands of the correct molecular weight for both groups. However, probing for phosphorylated Syk yields no visible bands. Syk is only expressed in the B lymphocyte population within the PBMCs, and only a small proportion of B cells will show activation of Syk after treatment. Therefore, the effect of the IgM treatment is diluted within the protein lysate and undetectable when analyzed by WB. When doing the same analysis by flow cytometry, co-staining with CD19 and CD4, B and T cell-specific markers, respectively, can identify B cells. CD19 positive and CD4 negative B cells can be examined for Syk phosphorylation. As expected, there is no activation of Syk in untreated cells. However about 10% of B cells become positive for phospho-Syk following IgM treatment. Thus flow cytometry, unlike western blotting has the capacity to identify and resolve this small but real sub-population of B lymphocytes from a heterogeneous pool of human PBMCs.

Flow Cytometry Validated Antibodies for CyTOF®

CyTOF® mass cytometry single cell proteomics platform uses metal conjugated antibodies to simultaneously profile over 30 proteins within a single cell.

Cell Signaling Technology (CST) offers a diverse portfolio of thoroughly validated flow cytometry antibodies in PBS formulation, suitable for use with the CyTOF® mass cytometer.

  • PBS-formulated CST™ antibodies are ideal for conjugating to metals for use with the CyTOF® instrument.
  • High concentration antibody formulations (> 1mg/ml) yield improved conjugation success.
  • Choose from hundreds of flow cytometry validated CST™ antibodies.