Fix & Perm

At the beginning of 2014 AN DER GRUB has been acquired by Nordic-MUbio. All products from AN DER GRUB are still available from dianova under the new label of Nordic-MUbio. This also includes ADG’s products FIX & PERM® and ADG-Lyse.
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 acute 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® Kits and Reagents

Ordering No.DescriptionQuantity
GAS-002FIX & PERM Kit (CE-IVD)200 Tests
GAS-002-1FIX & PERM Kit (CE-IVD)1000 Tests
GAS-002A-1FIX & PERM Reagenz A (Fixing) (CE-IVD)1000 Tests
GAS-002B-1FIX & PERM Reagenz B (Permeabilisation) (CE-IVD)1000 Tests
GAS-002FOCFIX & PERM Kit (CE-IVD)free samples (15 Tests)
GAS-002MFIX & PERM Kit (CE-IVD)50 Tests

FIX & PERM® advantages

  • 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 antibodies for safe diagnostics of acute leukemia 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

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.

Especially in the diagnosis of acute leukemia, in cases lacking expression of B cell, T cell or myeloid surface antigens or disclosing an equivocal or ambiguous immunophenotype or in biphenotypic acute leukemia, the additional detection of cytoplasmic antigens for establishing proper lineage affiliation has been proven to be very important. Nordic MUbio offers a selection of markers especially optimized for the use with FIX & PERM® in leukemia diagnosis:

FITC and PE conjugated markers optimized for use with FIX & PERM® in leukemia diagnostics for simultaneous intra- and extracellular staining
Cat. No.AntigenIsotypeCloneFormatQuantity
GM-4011CD3IgG1UCHT1purified0,2 mg
GM-4012CD3IgG1UCHT1FITC-labeled100 Tests
GM-4013CD3IgG1UCHT1PE-labeled100 Tests
GM-4051CD22IgG1RFB4purified0,2 mg
GM-4052CD22IgG1RFB4FITC-labeled100 Tests
GM-4053CD22IgG1RFB4PE-labeled100 Tests
GM-4111LactoferrinIgG14C5purified0,2 mg
GM-4113LactoferrinIgG14C5PE-labeled100 Tests
GM-4131LysozymeIgG1LZ-2purified0,2 mg
GM-4132LysozymeIgG1LZ-2FITC-labeled100 Tests
GM-4152CD68IgG1Ki-M7FITC-labeled100 Tests
GM-4191Myeloperoxidase(MPO-C2)IgG18E6purified0,2 mg
GM-4192Myeloperoxidase(MPO-C2)IgG18E6FITC-labeled100 Tests
GM-4193Myeloperoxidase(MPO-C2)IgG18E6PE-labeled100 Tests
Combi panels optimized for FIX & PERM® for dual staining in leukemia diagnostics
Cat. No.AntigenCloneIsotypeFormatQuantity
GIC-201NegativkontrolleVl-AP / Vl-APIgG1 / IgG1FITC-labeled / PE-labeled50 Tests
GIC-206Lysozyme / LactoferrinLZ-2 / 4C5IgG1 / IgG1FITC-labeled / PE-labeled50 Tests
GIC-212Myeloperoxidase / Lactoferrin8E6 / 4C5IgG1 / IgG1FITC-labeled / PE-labeled50 Tests
GIC-213Myeloperoxidase / CD38E6 / UCHT1IgG1 / IgG1FITC-labeled / PE-labeled50 Tests
GIC-214Myeloperoxidase / CD228E6 / RFB4IgG1 / IgG1FITC-labeled / PE-labeled50 Tests