FIX&PERM®Cell Fixation and Permeabilization Kit

Intracellular staining of protein biomarkers using antibodies in flow cytometry has opened diverse new pathways for phenotypic and functional characterization of cells in both research and clinical diagnosis.

Definitive phenotypic identification of certain cell types can require labeling of both cell surface and intracellular markers. Additionally, the cytoplasmic localization of typical membrane molecules can prove to be a most reliable lineage marker, such as cytoplasmic CD3 and CD22 in undifferentiated leukemia.

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

FIX&PERM® is a simple flow cytometry cell preparation 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-labeled antibodies, allowing staining of extracellular surface markers and intracellular structures such as cytoplasmic enzymes, nuclear proteins, oncoproteins, cytokines and immunoglobulins.

FIX&PERM® Kit Advantages

• Mildly fixes cells, preserving their flow cytometric scatter characteristics
• Allows simultaneous characterization 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
• Proven technology – initially characterized in Knapp et al. in 1994, independently validated in studies such as Groeneveld et al. in 1996 and Lanza et al. in 1997, and cited in hundreds of peer-reviewed publications since
• Stringent QC procedures – the quality of each lot is confirmed using well-defined blood samples and subsequent comparison of scatter characteristics and immunostaining patterns, ensuring consistent and reliable results each time
• Stable – our formulations do not contain volatile organic solvents, enabling storage at ambient temperature during shipment and in laboratories
• Flexible – can be used with most monoclonal antibody conjugates, as well as our range of antibodies with optimized protocols for use with FIX&PERM®
• CE/IVD registered kits available for European customers in a clinical laboratory setting (contact us for further information)

FIX&PERM® Procedure

For each sample to be analyzed 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
• Analyze fixed cells within 24 hours

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

Furthermore, a number of Combi panels are offered for dual immunostaining protocols.

Scicons is now a part of Nordic-MUbio. We manufacture three mouse monoclonal antibodies that bind to double-stranded RNA molecules: their anti-dsRNA antibodies can be used as a diagnostic tool to detect whether an unknown pathogen is bacterial or viral in nature, including detection in paraffin-embedded fixed tissue samples.

The hybridomas that produce the J2, K1 and K2 antibodies were created in 1990, and their specificity, their binding to a range of synthetic double stranded polyribonucleotides and their lack of reaction with DNA and single stranded RNA has been extensively characterised (Schönborn et al., 1991).

J2 monoclonal antibody (mAb), mouse, IgG2a, kappa chain

The J2 anti-dsRNA IgG2a monoclonal antibody has become the Gold Standard in dsRNA detection. It was used initially for the study of plant viruses, but since the seminal paper of Weber et al. in 2006, where J2 was used to show that all the positive strand RNA viruses tested produced copious amounts of dsRNA in infected cells, this antibody has been used extensively in a wide range of systems, as documented in over 200 scientific publications.

J2 can be used to detect dsRNA intermediates of viruses as diverse as Hepatitis C virus, Dengue virus, rhinovirus, Chikungunya virus, Rabies virus, Polio virus, Classic swine fever virus, Brome mosaic virus and many more in cultured cells and also in fixed paraffin-embedded histolog-ical samples.

J2 has been used to elucidate how anti-viral responses are initiated, what counter-strategies viruses have adopted to avoid them, and to explore the viral life cylce by enabling ultrastructiural localisation studies of viral nucleic acid replication sites (Welsch et al., 2009 & Knoops et al., 2011).

J2 has been used successfully in electron microscopy, in immunofluorescence microscopy, in immunohistochemistry, and various immuno-capture methods, such as dot blots and ELISA. J2 has also been recommended as a diagnostic tool to detect whether an unkown pathogen is bacterial or viral in nature (Richardson et al., 2010). Recently J2 has also been used to monitor the removal of dsRNA from in vitro synthesized mRNA preparations that may have potential use in gene therapy (Kariko et al., 2011).

J2 monoclonal antibody (mAb), mouse, IgG2a, kappa chain

The K1 IgG2a monoclonal antibody recognises dsRNA with similar affinity to our widely used J2 antibody and is also sold in lyophilised form. It can be used for the histological and cytological detection of dsRNA in cells and tissues.

It has proven especially useful as an alternative to J2 to resolve cross-reactions and/or remove unwanted background, in those rare experi-mental setups where J2 did not provide satisfactory results.

K1 can be used to detect dsRNA intermediates of viruses as diverse as Hepatitis virus, Theiler’s murine encephalomyelitis virus or Japanese encephalitis virus. It has been for the detection of dsRNA in cultured cells and in fixed paraffin-embedded histological samples (see publications).

If Poly I:C needs to be detected we highly recommend using K1 rather than J2 because K1 has a much higher affinity for this synthetic polyribonucleotide (see Schönborn et al. 1991, Fig. 2).

K1 has been used successfully in immunofluorescence microscopy, in flow cytometry (FACS) and in immunocapture methods (such as dot-blot and ELISA).

J2 monoclonal antibody (mAb), mouse, IgG2a, kappa chain

K2 anti-dsRNA monoclonal antibody has an IgM isotype.

K2 is mainly used for specialised applications, where J2 and K1 – antibodies that can be shipped at ambient temperature – do not suffice. Due to its IgM isotype K2 is more prone to aggregation at high concentration, and therefore cannot be lyophilised and needs to be shipped on ice.

K2 has primarily been used in ELISA and sandwich ELISA. Generally, K2 can be used in applications where an anti-dsRNA antibody with an isotype other than IgG (IgG2a) is required.