Intracellular Flow

Intracellular flow cytometry enables the detection of transcription factors and associated proteins within heterogeneous cell populations.

The simultaneous analysis of multiple markers allows for the determination of critical time points, markers, and frequencies of cells moving along a particular differentiation pathway.

Transcription factors are proteins that bind to DNA and other proteins to regulate gene expression. They play key roles in cellular development and differentiation. Examples include FoxP3 for Treg differentiation and Sox17 for definitive endoderm.


FoxP3 is considered to be the master regulator of Tregs. Like many transcription factors, FoxP3 binds to thousands of genes, resulting in the up- or down-regulation of gene expression necessary for Treg function.1 SATB1, a genome organizer, is repressed by FoxP3, preventing the induction of T-effector cytokines, including IL-4 and IFN-γ.2

Similarly to intracellular cytokine staining, transcription factor detection using flow cytometry requires cellular fixation and permeabilization. Transcription factors are typically located in the nucleus bound to DNA and other proteins. Depending on the nature of the target molecule epitopes, different fixation and permeabilization buffers might be necessary.

The fixation and permeabilization of cells can compromise cell surface marker staining, which makes the choice of compatible buffers even more critical. To further enable easy transcription factor detection, BD has developed the BD Pharmingen transcription factor buffer set. This buffer system is strong enough to allow access to most intracellular antigens while maintaining the detectability of most cell surface markers.


Detection of human Tregs by intracellular flow cytometry.

Detection of human Tregs by intracellular flow cytometry

Human PBMCs were stained for surface markers using the following fluorescent antibodies: CD4 FITC, CD25 Brilliant Violet 421, and CD127 Alexa Fluor® 647. After washing, the cells were fixed and permeabilized using the BD Pharmingen transcription factor buffer set. Cells were then stained intracellularly with FoxP3 PE-CF594, washed, and acquired on a BD FACSVerse flow cytometer equipped with a PE-CF594 optional filter mirror unit. Gated Tregs (colored blue) have a CD4+ CD25high CD127dim FoxP3+ phenotype.

Detection of human Tregs by intracellular flow cytometry

Human PBMCs were stained for surface markers using the following fluorescent antibodies: CD4 FITC, CD25 Brilliant Violet 421, and CD127 Alexa Fluor® 647. After washing, the cells were fixed and permeabilized using the BD Pharmingen transcription factor buffer set. Cells were then stained intracellularly with FoxP3 PE-CF594, washed, and acquired on a BD FACSVerse flow cytometer equipped with a PE-CF594 optional filter mirror unit. Gated Tregs (colored blue) have a CD4+ CD25high CD127dim FoxP3+ phenotype.


Convenient Kits for the Study of Stem Cell Differentiation

As pluripotent stem cells differentiate into different cell types, expression of transcription factors and other proteins changes. In mammalian embryonic development, the definitive endoderm generates the liver, pancreas, and intestine.3-5 During specification into definitive endoderm, levels of the transcription factors Sox17 and FoxA2 and the cell surface marker CD184 (CXCR4) increase, while pluripotency markers such as Nanog and Sox2 decrease.6

Multicolor flow cytometry is an excellent method for determining the relative numbers of cells expressing markers of interest. This is useful for the study of cell differentiation pathways and specifically for the optimization, quantification, and comparison of differentiation protocols and the differentiation potentials of different cells..

To facilitate the study of transcription factors in stem cells, BD has developed several kits for the detection of key stemcell– specific transcription factors including the BD Stemflow™ human pluripotent stem cell transcription factor analysis kit (Cat. No. 560589), the BD Stemflow™ mouse pluripotent stem cell transcription factor analysis kit (Cat. No. 560585), the BD Stemflow™ human neural cell lineage analysis kit (Cat. No. 561526), and the BD Stemflow™ human definitive and pancreatic endoderm analysis kit (Cat. No. 562496). These kits contain optimized antibodies and buffer systems to characterize pluripotent stem cells and allow tracking of the differentiation of pluripotent stem cells into respective lineages.


Detection of human Tregs by intracellular flow cytometry.

Definitive endoderm differentiation of H9 hESCs

H9 human embryonic stem cells (hESCs) (WiCell, Madison, WI) were differentiated to definitive endoderm for three days according to a protocol in D’Amour et al.6 Differentiated cells were analyzed by flow cytometry using components of the BD Stemflow human definitive and pancreatic endoderm analysis kit and CD184 PE. As cells differentiated, levels of the pluripotency marker Nanog decreased, and levels of definitive endoderm markers Sox17 (green) and CD184 increased. Flow cytometry was performed on a BD™ LSR II flow cytometry system.

Definitive endoderm differentiation of H9 hESCs

H9 human embryonic stem cells (hESCs) (WiCell, Madison, WI) were differentiated to definitive endoderm for three days according to a protocol in D’Amour et al.6 Differentiated cells were analyzed by flow cytometry using components of the BD Stemflow human definitive and pancreatic endoderm analysis kit and CD184 PE. As cells differentiated, levels of the pluripotency marker Nanog decreased, and levels of definitive endoderm markers Sox17 (green) and CD184 increased. Flow cytometry was performed on a BD™ LSR II flow cytometry system.


1 Birzele F, Fauti T, Stahl H, et al. Next-generation insights into regulatory T cells: expression profiling and FoxP3 occupancy in Human. Nucleic Acids Res. 2011;39:7946-7960.

2 Beyer M, Thabet Y, Müller RU, et al. Repression of the genome organizer SATB1 in regulatory T cells is required for suppressive function and inhibition of effector differentiation. Nat Immunol. 2011;12:898-907.

3 Wang P, McKnight KD, Wong DJ, et al. A molecular signature for purified definitive endoderm guides differentiation and isolation of endoderm from mouse and human embryonic stem cells. Stem Cells Dev. 2012;2:2273-2287.

4 Takayama K, Inamura M, Kawabata K, et al. Efficient and directive generation of two distinct endoderm lineages from human ESCs and iPSCs by differentiation stage-specific SOX17 transduction. PLoS One. 2011;6:e21780.

5 Murry CE, Keller G. Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development. Cell. 2008;132:661-680.

6 D’Amour KA, Agulnick AD, Eliazer S, Kelly OG, Kroon E, Baetge EE. Efficient differentiation of human embryonic stem cells to definitive endoderm. Nat Biotechnol. 2005; 23:1534-1541.