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FIX&PERM Cell Fixation and Permeabilization Kit

제품 소개

유세포분석 (flow cytometry)에서 항체를 사용한 단백질 biomarker의 세포내 염색은 연구나 진단 분야 모두에서 세포의 표현형 이나 기능을 파악하는데 새로운 옵션을 제공해 주는 방법입니다.

특정 세포 유형을 확실하게 식별하기 위해서는 세포 표면 및 세포내 마커 모두에 표지를 해야합니다.
실제로 FIX&PERM kit는 다양한 생물학적 검체 (혈액, 골수 등)에서 추출된 정상 및 악성 백혈구 집단 (leukocyte populations)의 분석에 적합합니다.

이처럼 많은 세포의 생물학적 및 기능적 연구를 위해 intracellular immunostaining이 필요합니다.
이를 위해 Nordic-MUbio/Exalpha는 동급 최고의 Cell Fixation and Permeabilization Kit를 제공합니다.

이 키트에는 Fixation Medium(시약 A) 및 Permeabilization Medium(시약 B)의 2가지 시약이 포함되어 있습니다.
먼저 시약 A로 현탁된 세포를 고정한 다음 시약 B로 세포막을 투과 시키게 됩니다.

이는 항체가 세포 내 구조에 접근할 수 있도록 하고 세포의 형태학적 산란 특성을 그대로 유지하게 합니다.

이 과정에서 특정 조성으로 만들어진 키트는 background를 줄이고 투과성 배지와 형광 색소 표지 항체를 동시에 추가할 수 있어,
cytoplasmic or nuclear enzymes, oncoproteins, cytokines, immunoglobulin 등과 같은 세포내 구조를 염색할 수 있습니다.

제품 특징

• flow cytometric scatter characteristics를 보존하면서 세포를 부드럽게 고정.
• intracellular and cell surface markers를 동시에 characterization.
• 모든 과정을 1시간 이내에 수행할 수 있으며, 즉시 분석하거나 24시간 동안 저장 가능
• 엄격한 QC 절차로 신뢰할 수 있는 결과 보장
• 최적화된 프로토콜을 가진 다양한 intracellular antibodies에 사용

• Storage: FIX&PERM® Cell Fixation and Permeabilization Kit reagents should be stored and used at room temperature. Do not freeze.
• Caution: This product is intended FOR RESEARCH USE ONLY, and FOR TESTS IN VITRO, not for use in diagnostic or therapeutic procedures involving humans or animals.

• BRAND: Nordic, Exalpha, Gallus, MUbio, Biologo, ADG, ELTI, Scicons, Bii

Fixation, permeabilization, and staining procedure:

For each sample to be analyzed using 50 µl of whole blood, bone marrow, or mononuclear cell suspension in a 5 ml tube

1. Add 100 µl of Reagent A (Fixation Medium, stored and used at room temperature)
2. Incubate for 15 minutes at room temperature
3. Add 5 ml phosphate-buffered saline and centrifuge cells for 5 minutes at 300 g
4. Remove supernatant and add to cell pellet 100 µl Reagent B and 20 µl of the appropriate monoclonal antibody\
5. Vortex at low speed for 1-2 seconds
6. Incubate for 15 minutes at room temperature
7. Wash cells with PBS as above
8. Remove supernatant and resuspend cells in sheath fluid for immediate
Analysis
9. Alternatively, resuspend cells in 0.5 ml 1.0 % formaldehyde and store them at 2-8°C in the dark. Analyze cells within 24 hours

Figure 1: Flow cytometric analysis of normal bone marrow (BM) after fixation and permeabilization using GAS-002, followed by immunostaining for lactoferrin and MPO-C2.

Figure 2: Flow cytometric analysis of bone marrow (BM) from a leukemia patient after fixation and pemeabilization using GAS-002, followed by immunostaining for lactoferrin and MPO-C2.

 Figure 3: Immunostaining with Nordic-MUbio CD22-PE conjugate of undifferentiated leukemia cells of B-ALL type.

Figure 4: Immunostaining with Nordic-MUbio CD3-PE conjugate of surface CD3-negative undifferentiated leukemia cells of T-ALL type.

Figure 5: Immunostaining with Nordic-MUbio anti-MPO-FITC conjugate of undifferentiated leukemia cells of AML type.

Figure 6: Forward and side scatter pattern of a normal blood sample treated with GAS-002.

 Figure 7: Double labeling of a normal blood sample treated with GAS-002, and immunostained for Lactoferrin (PE) and MPO-C2 (FITC).

Figure 8; Double labeling of a normal blood sample treated with GAS-002, and immunostained for CD3 (PE) and MPO-C2 (FITC).

Monoclonal antibodies optimized for use with FIX&PERM

Antigen Clone Isotype Format Quantity Cat. No.
CD3 UCHT1 IgG1 Purified 0.2 mg GM-4011
FITC-labeled 100 tests GM-4012
PE-labeled 100 tests GM-4013
CD22 RFB4 IgG1 Purified 0.2 mg GM-4051
FITC-labeled 100 tests GM-4052
PE-labeled 100 tests GM-4053
Lactoferrin 4C5 IgG1 Purified 0.2 mg GM-4111
PE-labeled 100 tests GM-4113
Myeloperoxidase
(MPO-C2)
8E6 IgG1 Purified 0.2 mg GM-4191
FITC-labeled 100 tests GM-4192
PE-labeled 100 tests GM-4193
Lysozyme LZ-2 IgG1 Purified 0.2 mg GM-4131
FITC-labeled 100 tests GM-4132
CD68 Ki-M7 IgG1 FITC-labeled 100 tests GM-4152
IL-1 beta FIB3 IgG1 FITC-labeled 100 tests GM-4162
IFN-gamma GZ4 IgG1 FITC-labeled 100 tests GM-4172


Furthermore, a number of Combi panels are offered for dual immunostaining protocols.

Antigen Clone Isotype Format Quantity Cat. No.
Negative control Vl-AP IgG1 FITC-labeled 50 tests GIC-201
Vl-AP IgG1 PE-labeled
Lysozyme LZ-2 IgG1 FITC-labeled 50 tests GIC-206
Lactoferrin 4C5 IgG1 PE-labeled
Myeloperoxidase 8E6 IgG1 FITC-labeled 50 tests GIC-212
Lactoferrin 4C5 IgG1 PE-labeled
Myeloperoxidase 8E6 IgG1 FITC-labeled 50 tests GIC-213
CD3 UCHT1 IgG1 PE-labeled
Myeloperoxidase 8E6 IgG1 FITC-labeled 50 tests GIC-214
CD22 RFB4 IgG1 PE-labeled

References

Recent application paper:

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.

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.
Cytometry B Clin Cytom. 2020 Aug 28. doi: 10.1002/cyto.b.21946. Online ahead of print.

Further reading:

– Gerna, G., Percivalle, E., Lilleri, D., Lozza, L., Fornara, C., Hahn, G., Baldanti, F. & Revello, M. G. (2005) J Gen Virol 86, 275-84.
– Groeneveld, K., te Marvelde, J. G., van den Beemd, M. W., Hooijkaas, H. & van Dongen, J. J. (1996) Leukemia 10, 1383-9.
– Haranaga, S., Yamaguchi, H., Friedman, H., Izumi, S., & Yamamoto, Y. (2001) Infect Immun 69, 7753-9.
– Hegazy, A. N. & Klein, C. (2008) Leukemia 22, 2070-9.
– Kappelmayer, J., Gratama, J. W., Karaszi, E., Menendez, P., Ciudad, J., Rivas, R. & Orfao, A. (2000) J Immunol Methods 242, 53-65.
– 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
– Knapp, W., Majdic, O. & Strobl, H. (1993) Recent Results Cancer Res 131, 31-40.
– Knapp, W., Strobl, H. & Majdic, O. (1994) Cytometry 18, 187-98.
– Knapp, W., Strobl, H., Scheinecker, C., Bello-Fernandez, C. & Majdic, O. (1995) Ann Hematol 70, 281-96.
– Konikova, E., Glasova, M., Kusenda, J. & Babusikova, O. (1998) Neoplasma 45, 282-91.
– Lanza, F., Latorraca, A., Moretti, S., Castagnari, B., Ferrari, L. & Castoldi, G. (1997) Cytometry 30, 134-44.
– Millard, I., Degrave, E., Philippe, M. & Gala, J. L. (1998) Clin Chem 44, 2320-30.
– Nakase, K., Sartor, M. & Bradstock (1998) Cytometry 34, 198-202.
– 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
– Pickl, W. F., Majdic, O., Kohl, P., Stockl, J., Riedl, E., Scheinecker, C., Bello-Fernandez, C. & Knapp, W. (1996) J Immunol 157, 3850-9.
– Riera-Sans, L., & Behrens, A. (2007) J Immunol 178, 5690-700
– 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
– Sargent, R. L., Craig, F. E. & Swerdlow, S. H. (2009) Int J Clin Exp Pathol 2, 574-82
– Scheinecker, C., Strobl, H., Fritsch, G., Csmarits, B., Krieger, O., Majdic, O. & Knapp, W. (1995) Blood 86, 4115-23.
– Sedlmayr, P., Grosshaupt, B. & Muntean, W. (1996) Cytometry 23, 284-9.
– Strobl, H. & Knapp, W. (2004) J Biol Regul Homeost Agents 18, 335-9.
– Strobl, H., Scheinecker, C., Csmarits, B., Majdic, O. & Knapp, W. (1995) Br J Haematol 90, 774-82.
– Strobl, H., Scheinecker, C., Riedl, E., Csmarits, B., Bello-Fernandez, C., Pickl, W. F., Majdic, O. & Knapp, W. (1998) J Immunol 161, 740-8.
– Strobl, H., Takimoto, M., Majdic, O., Fritsch, G., Scheinecker, C., Hocker, P. & Knapp, W. (1993) Blood 82, 2069-78.
– Wang, X., Chang, X., Facchinetti, V., Zhuang, Y. & Su, B. (2009) J Immunol 182, 3597-608
-Fan J, Tang X, Wang Q, Zhang Z, Wu S, Li W, Liu S, Yao G, Chen H and Sun L, Mesenchymal stem cells alleviate experimental autoimmune cholangitis through immunosuppression and cytoprotective function mediated by galectin-9, Stem Cell Research & Therapy 2018, https://doi.org/10.1186/s13287-018-0979-x

Bingyu Shi, Jingjing Qi, Genhong Yao, Ruihai Feng, Zhuoya Zhang, Dandan Wang, Chen Chen, Xiaojun Tang, Liwei Lu, Wanjun Chen and Lingyun Sun
Mesenchymal stem cell transplantation ameliorates Sjögren’s syndrome via suppressing IL-12 production by dendritic cells.
Stem Cell Research & Therapy, 2018 https://doi.org/10.1186/s13287-018-1023

Rossella Zanin, Silvia Pegoraro, Gloria Ros, Yari Ciani, Silvano Piazza, Fleur Bossi, Roberta Bulla, Cristina Zennaro, Federica Tonon, Dejan Lazarevic, Elia Stupka, Riccardo Sgarra & Guidalberto Manfioletti HMGA1 promotes breast cancer angiogenesis supporting the stability, nuclear localization and transcriptional activity of FOXM1. Journal of Experimental & Clinical Cancer Research volume 38, Article number: 313 (2019)
https://doi.org/10.1186/s13046-019-1307-8

Manabu Itoh, Yosuke Mukae, Takahiro Kitsuka, Kenichi Arai, Anna Nakamura, Kazuyoshi Uchihashi, Shuji Toda, Kumika Matsubayashi, Jun-ichi Oyama, Koichi Node, Daisuke Kami, Satoshi Gojo, Shigeki Morita, Takahiro Nishida, Koichi Nakayama & Eiji Kobayashi, Development of an immunodeficient pig model allowing long-term accommodation of artificial human vascular tubes. Nature Communications volume 10, Article number: 2244 (2019) https://doi.org/10.1038/s41467-019-10107-1

주문정보

Product Code Product Name Product Type
GAS-002 FIX&PERM® Cell Fixation and Permeabilization Kit (CE) Buffers and Reagents
GM-4192 Mouse anti-Myeloperoxidase-C2 (MPO-C2), conjugated to FITC Primary Antibodies
GAS-003 NM-LYSE: Flow Cytometry Lysing Solution Buffers and Reagents
GIC-212 COMBI IC Reagent: Mouse anti-Myeloperoxidase-C2 (FITC) and Mouse anti Lactoferrin (PE) Primary Antibodies
GM-4132 Mouse anti Lysozyme, conjugated to FITC Primary Antibodies
GM-4113 Mouse anti-Lactoferrin, conjugated to PE Primary Antibodies

관련정보

제품 자료 FIXampPERM Cell Fixation and Permeabilization Kit.pdf Upload date: 2022.01.06
제품 MSDS MSDS-GAS-002.pdf Upload date: 2022.01.06