FIX,PERM® Kit 1000

Flow cytometric analyses with monoclonal antibodies were so far mainly restricted to cell surface molecules. Intracellular structures such as cytoplasmic or nuclear enzymes, oncoproteins, cytokines, immunoglobulins etc. were largely excluded from such studies. Also excluded from flow cytometric studies were cytoplasmic localizations of well-established membrane molecules like CD3 and CD22, which, in their cytoplasmic form, are the most reliable lineage markers in undifferentiated leukemia. With the FIX,PERM® Kit flow cytometric analysis of intracellular antigens has become as easy as surface antigen studies. The only prerequisite is the availability of suitable antibody conjugates. Most of the available monoclonal antibody conjugates can be used with the FIX,PERM® Kit, some determinants are sensitive, however, to the fixation step involved. This and the optimal fixation time have to be tested for each reagent._x000D_ Results must be interpreted by a certified professional before final interpretation. Analyses performed with this antibody should be paralleled by positive and negative controls. If unexpected results are obtained which cannot be attributed to differences in laboratory procedures, please contact us.

Buffers and Reagents

Flow Cytometry

Immunology, Oncology

Biological fluids (blood, bone marrow, and others) must be collected under sterile conditions. Anticoagulation with EDTA or heparin is recommended. The samples should be stored at room temperature until used. For optimal results, samples should be processed and analyzed within 24 hours. Samples with high numbers of non-viable cells might cause false results, such cases require determination of cell viability with e.g. propidium iodide. All biological samples have to be handled with caution. Always consider them as potentially infective. Use appropriate precautions such as gloves, lab-coat, etc._x000D_ _x000D_ Fixation, permeabilization and staining procedure:_x000D_ _x000D_ • For each sample to be analyzed add 50 µL of whole blood, bone marrow or mononuclear cell suspension in a 5mL tube_x000D_ • Add 100 µL of Reagent A (Fixation Medium, stored and used at room temperature)_x000D_ • Incubate for 15 minutes at room temperature_x000D_ • Add 5mL phosphate buffered saline and centrifµge cells for 5 minutes at 300 g_x000D_ • Remove supernatant and add to cell pellet 100 µL Reagent B (Permeabilization Medium) and 20 µL of the appropriate Nordic-MUbio monoclonal antibody conjµgate_x000D_ • Vortex at low speed for 1-2 seconds_x000D_ • Incubate for 15 minutes at room temperature_x000D_ • Wash cells with phosphate buffered saline as described above_x000D_ • Remove supernatant and resuspend cells in sheath fluid for immediate analysis or resuspend cells in 0.5 mL 1.0 % formaldehyde and store them at 2-8°C in the dark. Analyze fixed cells within 24 hours._x000D_ _x000D_ _x000D_ Comments: Special cases (diluted bone marrow samples, other samples containing low soluble protein) might benefit from replenishment with plasma components before the FIX,PERM® treatment in order to create a milieu, which more closely resembles the stuation in anti-coagulated blood. For that pupose addition of IgG preparations (e.g. Beriglobulin P, ZLB Behring, final concentration 10mg/mL) and Human serum albumin (e.g. Human albumin “Behring” 20% - infusion solution, final concentration 40mg/mL) is recommended.

This FIX,PERM® Cell Fixation and Permeabilization Kit contains 2 reagents: Fixation Medium (Reagent A) and Permeabilization Medium (Reagent B); It is intended for first fixing cells in suspension with Reagent A and then permeabilizing the cell membranes with Reagent B; This procedure gives antibodies access to intracellµLar structures and leaves the morphological scatter characteristics of cells intact. Specific formµLations reduce background staining and allow simµLtaneous addition of permeabilization medium and fluorochrome labeled antibodies; FIX,PERM® is suitable for the analysis of; Normal and malignant leukocyte popµLations derived from various Human biological samples (blood, bone marrow and others) using flow cytometry; ResµLts must be put within the context of other diagnostic tests as well as the clinical history of the patient by a certified professional before final interpretation._x000D_ _x000D_ Format: 1 x 100mL vial of Fixation Medium (Reagent A) and 1 x 100mL vial Permeabilization Medium (Reagent B). FIX,PERM® Reagents are designed for use with all commercially available flow cytometers. Alignment and compensation shoµLd be performed according to manufacturer´s instructions._x000D_ _x000D_ The quality of each FIX,PERM® Lot is determined by fixation and permeabilization of well defined blood samples from representative donors and subsequent comparison of forward and side scatter characteristics of obtained leukocytes, as well as immunostaining efficiency for several membranous and cytoplasmic antigens

For professional users only._x000D_ Reagent A of FIX&PERM® Cell Fixation and Permeabilization Kit contains fomaldehyde and is labelled: Harmful. Formaldehyde is toxic, allergenic and a suspected carcinogen. Never pipette by mouth and avoid contact with eyes, skin and clothing. Proper handling procedures are recommended. As a main rule, persons under 18 years of age are not allowed to work with this product. Users must be carefully instructed in the proper working procedure, the dangerous properties of the product and the necessary safety instructions. Please refer to the Material Safety Data Sheet (MSDS) for additional information._x000D_ Dispose product remainders according to local regulations.

Recent application papers:_x000D_ _x000D_ Mestrum SGC, de Wit NCJ, Drent RJM, Hopman AHN, Ramaekers FCS, Leers MPG.Proliferative activity is disturbed in myeloproliferative neoplasms (MPN), myelodysplastic syndrome (MDS), and MDS/MPN diseases. Differences between MDS and MDS/MPN._x000D_ Cytometry B Clin Cytom. 2021 May;100(3):322-330. doi: 10.1002/cyto.b.21946. _x000D_ _x000D_ Mestrum SGC, Cremers EMP, de Wit NCJ, Drent RJM, Ramaekers FCS, Hopman AHN, Leers MPG. Integration of the Ki-67 proliferation index into the Ogata score improves its diagnostic sensitivity for low-grade myelodysplastic syndromes. Leuk Res. 2022 Feb;113:106789. doi: 10.1016/j.leukres.2022.106789. _x000D_ _x000D_ Mestrum SGC, Cremers EMP, de Wit NCJ, Drent RJM, Ramaekers FCS, Hopman AHN, Leers MPG. Optimized gating strategy and supporting flow cytometry data for the determination of the Ki-67 proliferation index in the diagnosis of myelodysplastic syndrome. Data Brief. 2022 Feb 22;41:107976. doi: 10.1016/j.dib.2022.107976._x000D_ _x000D_ Nies KPH, Kraaijvanger R, Lindelauf KHK, Drent RJMR, Rutten RMJ, Ramaekers FCS, Leers MPG. Determination of the proliferative fractions in differentiating hematopoietic cell lineages of normal bone marrow. Cytometry A. (2018) 93, 1097-1105 . doi: 10.1002/cyto.a.23564_x000D_ _x000D_ _x000D_ Further reading:_x000D_ - Gerna, G., Percivalle, E., Lilleri, D., Lozza, L., Fornara, C., Hahn, G., Baldanti, F. , Revello, M. G. (2005) J Gen Virol 86, 275-84._x000D_ - Groeneveld, K., te Marvelde, J. G., van den Beemd, M. W., Hooijkaas, H. , van Dongen, J. J. (1996) Leukemia 10, 1383-9._x000D_ - Haranaga, S., Yamaguchi, H., Friedman, H., Izumi, S., , Yamamoto, Y. (2001) Infect Immun 69, 7753-9._x000D_ - Hegazy, A. N. , Klein, C. (2008) Leukemia 22, 2070-9._x000D_ - Kappelmayer, J., Gratama, J. W., Karaszi, E., Menendez, P., Ciudad, J., Rivas, R. , Orfao, A. (2000) J Immunol Methods 242, 53-65._x000D_ - Kline, M. P., Rajkumar, S. V., Timm, M. M., Kimlinger, T. K., Haug, J. L., Lust, J. A., Greipp, P. R. , Kumar, S. (2007) Leukemia 21, 1549-60_x000D_ - Knapp, W., Majdic, O. , Strobl, H. (1993) Recent Results Cancer Res 131, 31-40._x000D_ - Knapp, W., Strobl, H. , Majdic, O. (1994) Cytometry 18, 187-98._x000D_ - Knapp, W., Strobl, H., Scheinecker, C., Bello-Fernandez, C. , Majdic, O. (1995) Ann Hematol 70, 281-96._x000D_ - Konikova, E., Glasova, M., Kusenda, J. , Babusikova, O. (1998) Neoplasma 45, 282-91._x000D_ - Lanza, F., Latorraca, A., Moretti, S., Castagnari, B., Ferrari, L. , Castoldi, G. (1997) Cytometry 30, 134-44._x000D_ - Millard, I., Degrave, E., Philippe, M. , Gala, J. L. (1998) Clin Chem 44, 2320-30._x000D_ - Nakase, K., Sartor, M. , Bradstock (1998) Cytometry 34, 198-202._x000D_ - Pascale, F., Contreras, V., Bonneau, M., Courbet, A., Chilmonczyk, S., Bevilacqua, C., Epardaud, M., Niborski, V., Riffault, S., Balazuc, A. M., Foulon, E., Guzylack-Piriou, L., Riteau, B., Hope, J., Bertho, N., Charley, B. , Schwartz-Cornil, I. (2008) J Immunol 180, 5963-72_x000D_ - Pickl, W. F., Majdic, O., Kohl, P., Stockl, J., Riedl, E., Scheinecker, C., Bello-Fernandez, C. , Knapp, W. (1996) J Immunol 157, 3850-9._x000D_ - Riera-Sans, L., , Behrens, A. (2007) J Immunol 178, 5690-700_x000D_ - Roberts, J. L., Lengi, A., Brown, S. M., Chen, M., Zhou, Y. J., O'Shea, J. J. , Buckley, R. H. (2004) Blood 103, 2009-18_x000D_ - Sargent, R. L., Craig, F. E. , Swerdlow, S. H. (2009) Int J Clin Exp Pathol 2, 574-82_x000D_ - Scheinecker, C., Strobl, H., Fritsch, G., Csmarits, B., Krieger, O., Majdic, O. , Knapp, W. (1995) Blood 86, 4115-23._x000D_ - Sedlmayr, P., Grosshaupt, B. , Muntean, W. (1996) Cytometry 23, 284-9._x000D_ - Strobl, H. , Knapp, W. (2004) J Biol Regul Homeost Agents 18, 335-9._x000D_ - Strobl, H., Scheinecker, C., Csmarits, B., Majdic, O. , Knapp, W. (1995) Br J Haematol 90, 774-82._x000D_ - Strobl, H., Scheinecker, C., Riedl, E., Csmarits, B., Bello-Fernandez, C., Pickl, W. F., Majdic, O. , Knapp, W. (1998) J Immunol 161, 740-8._x000D_ - Strobl, H., Takimoto, M., Majdic, O., Fritsch, G., Scheinecker, C., Hocker, P. , Knapp, W. (1993) Blood 82, 2069-78._x000D_ - Wang, X., Chang, X., Facchinetti, V., Zhuang, Y. , Su, B. (2009) J Immunol 182, 3597-608

Ambient Temperature