SAMPLE PROCESSING
CELL FIXATION AND PERMEABILIZATION KIT
FEATURES
- 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®
PROCEDURE
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.
METHODOLOGY
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
