FIX & PERM® Cell Permeabilization Kit

Intracellular staining of protein biomarkers using antibodies in flow cytometry has opened up a diversity of new options for phenotypic and functional characterisation of cells in both research and clinical diagnosis.

Indeed, definitive phenotypic identification of certain cell types can require labelling of both cell surface and intracellular markers. Additionally, the cytoplasmic localisation of typical membrane molecules can prove to be a most reliable lineage marker, such as cytoplasmic CD3 and CD22 in undifferentiated leukaemia.

In such cases, and for many other cell biological and functional investigations, intracellular immunostaining is a necessity. For this purpose Nordic-MUbio offers a class-leading cell fixation and permeabilization kit – FIX&PERM® – which offers a number of advantages over other methods for intracellular staining of cells in flow cytometry.

FIX&PERM®

• Mildly fixes cells, preserving their flow cytometric scatter characteristics

• Allows simultaneous characterisation of both intracellular and cell surface markers

• Rapid technique – whole procedure can be carried out in less than one hour, ready for immediate analysis or storage for 24 hours

• Stringent QC procedures – the quality of each lot is determined using well-defined blood samples and subsequent comparison of scatter characteristics of obtained leukocyte populations, ensuring consistent and reliable results lot after lot

• A range of intracellular antibodies with optimised protocols for use with FIX&PERM®

FIX&PERM® is a simple procedure making use of two reagents. Reagent A gently fixes cells, while Reagent B permeabilizes them. The specific formulations reduce background and allow simultaneous addition of permeabilization medium and fluorochrome labelled antibodies, allowing staining of intracellular structures such as cytoplasmic or nuclear enzymes, oncoproteins, cytokines, immunoglobulin’s, etc.

Procedure

For each sample to be analysed use 50 µl of whole blood, bone marrow or mononuclear cell suspension in a 5 ml tube

• Add 100 µl of Reagent A (Fixation Medium, stored and used at room temperature)

• Incubate for 15 minutes at room temperature

• Add 5 ml phosphate buffered saline and centrifuge cells for 5 minutes at 300 g

• Remove supernatant and add to cell pellet 100 µl Reagent B (Permeabilization Medium) and 20 µl of the appropriate monoclonal antibody conjugate

• Vortex at low speed for 1-2 seconds

• Incubate for 15 minutes at room temperature

• Wash cells with phosphate buffered saline as described above

• Remove supernatant and resuspend cells in sheath fluid for immediate analysis or resuspend cells in 0.5 ml 1.0 % formaldehyde and store at 2-8°C in the dark

• Analyse fixed cells within 24 hours

Products optimised for use with FIX&PERM®

The majority of available monoclonal antibody conjugates can be used with FIX&PERM®. However, in some cases the fixation step may have to be optimised.

Nordic-MUbio offers a number of mouse anti-human conjugated (PE/FITC) monoclonal antibodies optimised for use with FIX&PERM®.