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Leucoperm

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BUF09C
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BUF09B
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BUF09
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Flow cytometric analyses with monoclonal antibodies have been restricted primarily to cell surface molecules. Intracellular structures such as cytoplasmic or nuclear enzymes, oncoproteins, cytokines, immunoglobulins etc. were largely excluded from such assays.

Also excluded from flow cytometric assays were cytoplasmic localizations 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
Regulatory
For research purposes only
Guarantee
12 months from date of despatch

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.

This product has been reported to work in the following applications. This information is derived from testing within our laboratories, peer-reviewed publications or personal communications from the originators. Please refer to references indicated for further information. For general protocol recommendations, please visit the antibody protocols page.
Application Name Verified Min Dilution Max Dilution
Flow Cytometry
LEUCOPERM reagents are intended for fixing cells in suspension with Reagent A and then permeabilizing the cells with Reagent B. The specific formulations reduce background staining and allow simultaneous addition of permeabilization medium and fluorochrome labeled antibodies.
Instructions For Use
BUF09C, BUF09B, BUF09
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. Re-suspend 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.

References for Leucoperm

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    J Nutr Biochem. 20: 254-60.
  2. Grundy, M. et al. (2010) The FLT3 internal tandem duplication mutation is a secondary target of the aurora B kinase inhibitor AZD1152-HQPA in acute myelogenous leukemia cells.
    Mol Cancer Ther. 9: 661-72.
  3. Taylor, L. et al. (2010) The effect of acute hypoxia on heat shock protein 72 expression and oxidative stress in vivo.
    Eur J Appl Physiol. 109 (5): 849-55.
  4. Bairey, O. et al. (2010) Arsenic-trioxide-induced apoptosis of chronic lymphocytic leukemia cells.
    Int J Lab Hematol. 32 (1 Pt 1): e77-85.
  5. Myles, A. et al. (2011) Expression of Toll-like receptors 2 and 4 is increased in peripheral blood and synovial fluid monocytes of patients with enthesitis-related arthritis subtype of juvenile idiopathic arthritis.
    Rheumatology (Oxford). 50: 481-8.
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    PLoS Negl Trop Dis. 5:e962.
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    J Allergy Clin Immunol. 137 (3): 955-7.e8.
  9. View The Latest Product References
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    Front Immunol. 8: 615.
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    Exp Ther Med. 14 (2): 1284-92.
  12. Jiang, W.J. et al. (2017) Structure-activity relationship of the inhibitory effects of flavonoids on nitric oxide production in RAW264.7 cells.
    Bioorg Med Chem. 25 (2): 779-788.
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  18. Martelli, P. et al. (2021) Immune B cell responsiveness to single-dose intradermal vaccination against Mycoplasma hyopneumoniae..
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BUF09C

150778 155353 157139 163682 166707

BUF09B

154793 157137 166036

BUF09

149239 157136 165465

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