Also excluded from flow cytometric studies were cytoplasmic localisations of well established membrane molecules such as CD3 and CD22.
LEUCOPERM reagents allow intracellular antigen analysis with the same ease as surface antigens. The only prerequisite is the availability of suitable antibody conjugates. Most commercially available monoclonal antibody conjugates can be used with LEUCOPERM reagents. Some determinants are sensitive, however, to the fixation step involved. This and the optimal fixation time may have to be determined experimentally for each antibody conjugate.
- Product Form
- Reagent A - Fixation medium
Reagent B - Permeabilisation medium
- Preservative Stabilisers
- Formaldehyde in Reagent A
- LEUCOPERM Cell Permeabilisation reagents should be stored and used at room temperature. DO NOT FREEZE. Do not use reagents if a precipitate forms or discolouration occurs.
- 12 months from date of despatch
- For research purposes only
Applications of Leucoperm
|Application Name||Verified||Min Dilution||Max Dilution|
LEUCOPERM reagents are intended for fixing cells in suspension with Reagent A and then permeabilising the cells with Reagent B. The specific formulations reduce background staining and allow simultaneous addition of permeabilisation medium and fluorochrome labelled antibodies.
- Instructions For Use
- For the detection of cell cycle antigens such as Ki-67, PCNA and BrdU, methanol modification is recommended - see protocol #F5.
1. Prepare cells in the appropriate manner. Adjust cell suspension to a concentration of 1 x 107 cells/ml in PBS/BSA. Whole blood samples may also be used. Bio-Rad recommend the use of EDTA anti-coagulant in these circumstances, although satisfactory results may be obtained using heparin or acid-citrate dextrose.
2. Add 100ul of cell suspension to the appropriate number of test tubes.
If required, perform staining of cell surface antigens at this stage. Following staining for the recommended period, wash cells once in PBS/BSA and discard supernatant.
3. Add 100ul of Reagent A (fixation medium, stored at room temperature).
4. Incubate for 15 minutes at room temperature.
5. Add 3ml PBS/BSA and centrifuge for 5 minutes at 300 x g. Remove supernatant.
6. Resuspend cells in 100ul of Reagent B (Permeabilization Medium).
7. Immediately add recommended volume of the appropriate directly conjugated antibody. Vortex and incubate for 30 minutes at room temperature.
If using an unconjugated primary antibody, wash in 3ml of PBS/BSA (as per step 5) and then repeat step 7 using an appropriate secondary antibody. There is no requirement to add further Leucoperm.
8. Wash once in PBS/BSA. Remove supernatant and resuspend cells in sheath fluid for immediate analysis or resuspend cells in 0.25ml of 0.5% formaldehyde and store them at 2-8oC in the dark. Analyse fixed cells within 24 hours.
Product Specific References
References for Leucoperm
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J Cell Biochem. Nov 25. [Epub ahead of print]
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J Allergy Clin Immunol. 137 (3): 955-7.e8.
Suradhat, S. et al. (2015) A novel DNA vaccine for reduction of PRRSV-induced negative immunomodulatory effects: A proof of concept.
Vaccine. 33 (32): 3997-4003.
Dishon, S. et al. (2017) Inhibition of Myeloid Differentiation Factor 88 Reduces Human and Mouse T-Cell Interleukin-17 and IFN&gamma Production and Ameliorates Experimental Autoimmune Encephalomyelitis Induced in Mice.
Front Immunol. 8: 615.
Nie, H. et al. (2017) Phenotypic switch in lung interstitial macrophage polarization in an ovalbumin-induced mouse model of asthma.
Exp Ther Med. 14 (2): 1284-92.