Although flow cytometry was originally developed to detect cell surface markers, it has become increasingly common for researchers to study intracellular targets in parallel. However, because detecting intracellular biomolecules requires that cells be fixed and permeabilized – a process that can prevent some antibodies from recognizing native cell surface antigens – it is often necessary to stain cell surface markers on live cells before performing a further round of immunostaining once cells have been fixed in order to detect intracellular targets. This not only adds extra steps to flow cytometry protocols, but it also increases the risk of error. To overcome this problem, we developed FIX&PERM® as a convenient solution to improve flow cytometry efficiency. Using FIX&PERM®, researchers have demonstrated that combining the intracellular marker Ki-67 with various CD markers increases its potential as a prognostic biomarker.
FIX&PERM® streamlines flow cytometry immunostaining
Our FIX&PERM® Cell Fixation and Permeabilization Kit enables simultaneous characterization of both cell surface markers and intracellular targets, reducing the duration of the entire flow cytometry immunostaining process to less than an hour. First, users incubate their cells with fluorochrome labeled antibodies to extracellular differentiation markers, then they fix their cells with reagent A. After this, they add labeled antibodies to intracellular proteins diluted in reagent B; this simultaneously permeabilizes the cells and allows the antibodies to bind their target antigens. Samples are then ready for immediate flow cytometric analysis or can be stored for 24 hours at 2-8°C in the dark.
|Product Code||Product Name||Product Type|
|GAS-002||FIX&PERM® Cell Fixation and Permeabilization Kit (CE)||Buffers and Reagents|
|GM-4192||Mouse anti Myeloperoxidase-C2 (MPO-C2), conjugated to FITC||Primary Antibodies|
|GAS-003||NM-LYSE: Flow Cytometry Lysing Solution||Buffers and Reagents|
|GIC-212||COMBI IC Reagent: Mouse anti Myeloperoxidase-C2 (FITC) and Mouse anti Lactoferrin (PE)||Primary Antibodies|
|GM-4132||Mouse anti Lysozyme, conjugated to FITC||Primary Antibodies|
|GM-4113||Mouse anti Lactoferrin, conjugated to PE||Primary Antibodies|
Supporting flow cytometric analysis of Ki-67
The proliferation marker Ki-67 is widely used to predict the biological behavior of cancers and to assess how solid tumors will react to chemotherapy by means of its detection in tumor biopsies by IHC. Yet, to date, it has seen little use for flow cytometric analyses of cancers of the blood and blood-forming tissue (the bone marrow and lymphatic tissue). To investigate how the prognostic utility of Ki-67 might be further extended, Mestrum et al recently employed flow cytometry to monitor Ki-67 in bone marrow aspirates taken from patients suffering from three major categories of malignant myeloid bone marrow disorders. Using FIX&PERM® to enable extracellular staining of multiple CD markers in conjunction with intracellular staining of Ki-67, they showed that flow cytometric analysis of the proliferating (Ki-67 positive) cell fraction showed distinct differences between the disease states. This finding promises to help classify these malignancies to determine an appropriate form of treatment.