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Rapid and simple detection system of apoptotic cells, with simultaneous differentiation of necrotic cells. ROTITEST ® Annexin V ROTITEST ® BioAnalysis Grade, ready-to-use Mechanism During the first stages of apoptosis, phosphatidylserine (PS) is translocated from the inner membran layer to the outer surface of the cell. The ROTITEST ® Annexin V Kit uses the Ca 2+ dependent binding efficiency of Annexin V to PS in order to label cells with damaged cell membranes. Additionally, propidium iodide is used to counter stain nuclei of cells with opened membranes, hence those which undergo necrotic degradation. Thus, cells with yellow-green membrane staining only can be identified as apoptotic, while double stained cells (with yellow-green membranes plus orange-red nuclei) are classified as necrotic. Analysis is performed via flow cytometry or fluorescence microscopy. Simple, rapid application For adherent and suspension cells For cultured and primary cells (also for yeast cells) Discriminating between apoptotic and necrotic cells Result in approx. 30 minutes Figure 2: Fluorescence microscopic analysis of Jurkat cells following induction of apoptosis by staurosporine. Assayed using 10 5 cells in 100 µl Annexin binding buffer, 5 µl Annexin V-FITC conjugate and 5 µl propidium iodide solution, 15 min. RT. A) FITC fluorescence (green), B) propidium iodide fluorescence (red), C) bright field image, D) composite image. Apoptotic cells are FITC positive, PI negative. FITC pos/PI pos cells may be classified as being necrotic. Healthy cells are FITC/PI neg. Each package contains simple-to-follow instructions-for-use. The kit contains: Annexin V-FITC conjugate, propidium iodide solution, Annexin V binding buffer, detailed instructions-for-use. Excluding the columns, contents of this Kit may not be bought separately. Figure 1: Flow cytometry analysis of Jurkat cells. Assayed using 5 × 10 5 cells in 500 µl Annexin binding buffer, 25 µl Annexin V-FITC conjugate and 25 µl propidium iodide solution, 15 min. RT. FITC-A: FITC staining (binding of Annexin-FITC conjugate), PE-A: Propidium iodide staining A,B) Cells after induction of apoptosis by Staurosporin, under pure annexin-FITC staining (A) or under annexin-FITC / PI staining (B), respectively. C,D) Uninduced control cells under annexin-FITC / PI staining (C) and without staining (D), respectively. Due to the binding of Annexin V to PS on the outer membrane surface, apoptotic cells are Annexin-FITC positive, but PI negative (Q4, circle). Q3 displays healthy cells (neg/neg), Q2 necrotic cells (Annexin-FITC pos and PI pos). Cells in Q1 (PI pos/ Annexin-FITC neg) are dead, non-apoptotic cells.
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Sterile ROTI ® Stock 10x PBS 10x conc., BioScience Grade, ready-to-use, sterile filtered 10fold stock solution phosphate buffered saline, pH 7,4. Very versatile standard solution for detection systems in protein biochemistry, cytological stainings and molecular biological assays. Directions for use 1 x PBS is, for instance, used as basic solution for Western-Blot analyses, ELISAs and other enzymatic assays, for solving and dilution of proteins, for immunocytological and immunohistological assays, as well as for in situ hybridisations, apoptosis detection systems and staining of nuclei.
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