| Artikelnummer | GAS-002FOC |
|---|---|
| Artikelgruppe | Zellfixierung, Zellpermeabilisierung |
| Artikelbezeichnung | FIX & PERM Kit |
| Testprinzip | Erythrozytenlyse/Zellfixierung & -permeabilisierung, zur Färbung intrazellulärer Antigene für die Durchflusszytometrie |
| Inhalt | 1 x 5 ml Reagenz A (Fixierungsmedium), 1 x 5 ml Reagenz B (Permeabilisierungsmedium) |
| Zweckbestimmung | für In-vitro-Diagnostik |
| Packungsgröße | 15 Tests, Muster |
| Hersteller / Marke | Nordic-MUbio |
- GAS-002FOC – Datenblatt
- MSDS_FIX&PERM_EN-2019
ProductThis 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 intracellular structures and leaves the morphological scatter characteristics of cells intact. Specific formulations reduce background staining and allow simultaneous addition of permeabilization medium and fluorochrome labeled antibodies. FIX&PERM® is suitable for the analysis of normal and malignant leukocyte populations derived from various human biological samples (blood, bone marrow and others) using flow cytometry. Results 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.Format: 4 x 5ml vial of Fixation Medium (Reagent A) and 4 x 5ml vial Permeabilization Medium (Reagent B). FIX&PERM® Reagents are designed for use with all commercially available flow cytometers. Alignment and compensation should be performed according to manufacturer´s instructions.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 antigensFormulation: 4 x 5ml vial of Fixation Medium (Reagent A) and 4 x 5ml vial Permeabilization Medium (Reagent B). FIX&PERM Reagents are designed for use with all commercially available flow cytometers. Alignment and compensation should be performed according to manufacturer
ApplicationBiological 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.
Fixation, permeabilization and staining procedure:
- For each sample to be analyzed add 50 µl of whole blood, bone marrow or mononuclear cell suspension in a 5ml tube
- Add 100 µl of Reagent A (Fixation Medium, stored and used at room temperature)
- Incubate for 15 minutes at room temperature
- Add 5ml 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 Nordic-MUbio 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 them at 2-8°C in the dark. Analyze fixed cells within 24 hours.
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.
ReferencesRecent 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 Sep 3. doi: 10.1002/cyto.a.23564.
Here you can find more references and information about FIX&PERM.
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1.抗原性是指抗原与其所诱导产生的抗体或致敏淋巴细胞特异性结合的能力。抗原性的强弱与抗原分子的大小、化学成分、抗原决定簇的结构、抗原与被免疫动物亲缘关系的远近等有密切关系。通常认为抗原的分子量愈大、化学组成愈复杂、立体结构愈完整以及与被免疫动物的亲缘关系愈远，则抗原性愈强。
2.免疫原性是指能够刺激机体形成特异抗体或致敏淋巴细胞的能力。即指抗原能刺激特定的免疫细胞，使免疫细胞活化、增殖、分化，最终产生免疫效应物质抗体和致敏淋巴细胞的特性。也指抗原刺激机体后，机体免疫系统能形成抗体或致敏T淋巴细胞的特异性免疫反应。
可与MHCⅡ类分子结合的都是蛋白性抗原；多糖和脂类不易于MHCⅡ类分子连接，难以被TH细胞识别，因而多不是良好的免疫原；但有时可以诱导抗体性免疫应答。 抗原递呈（antigenpresentation）是辅佐细胞向辅助性T细胞展示抗原和MHCⅡ类分子的复合物，并使之与TCR结合的过程。这个过程是几乎所有淋巴细胞活化的必需步骤。抗原递呈之前，经处理后的抗原肽段已经连接在MHC分子顶端的槽中，这个复合物便是TCR的配体。TCR与配体结合的精确模式尚未清楚，一个合理的说法是TCR中α和β链的V段接触MHC分子的α螺旋（形成MHC分子顶端槽的肽段），使高可变的连接部（V-J及V-D-J）与抗原肽段相结合。这样保证了TCR识别抗原的特异性。
超抗原的递呈有独特的模式，它不需要胞内处理，可以直接与MHCⅡ类分子结合。超抗原不结合在MHCⅡ类分子的顶端槽中，而是结合在槽的外侧；与TCR结合时，不结合其α链，只结合β链的V节段。超抗原对TCR和MHCⅡ类分子的结合都非常牢固，象一支双向钩子将T细胞和辅佐细胞紧紧地连在一起，很容易使T细胞活化。另外，任何超抗原都只与含特殊β链V节段的TCR结合，这样的TCR约占外周T细胞总数的1%～10%，这一数字远远大于任何普通抗原所能识别的细胞数；所以某些产毒细胞感染时，容易发生急性期素休克综合征，就是超抗原刺激的结果。向左转|向右转
我们自身的细胞，蛋白也是抗原，但是自体在正常情况下没有针对自身的抗体。抗体产生的过程很复杂，我简单说一下：抗体由B细胞产生。本质是蛋白质。B细胞在成熟的过程中，编码抗体抗原识别区的那段基因会发生随机组合，这样就有可能会产生无数种抗体。但是如果一个B细胞产生了针对自身的抗体，那这个细胞就会和抚养它的细胞发生过于紧密的接触，被杀死。如果一个B细胞产生的抗体还不能识别任何抗原，那它就得不到足够的生长刺激，所以也不能存活。最后成熟的免疫细胞只占很小一部分。只有那些可以识别外源抗体的B细胞才能得到刺激，大量增殖。然后体内的抗体就增高了。
所以你的最后一句话不对。并不是人人都可以对任何抗原产生抗体的。比如青霉素过敏，过敏的人就是因为体内有针对青霉素的抗体IgE。而其他人就没有抗体。
