Human Caspase-3 FlowCytomix Simplex Kit

Transcription

1 PRODUCT INFORMATION & MANUAL Human Caspase-3 FlowCytomix Simplex Kit BMS82012FF For research use only. Not for diagnostic or therapeutic procedures. Human Caspase-3 FlowCytomix Simplex Kit North America Technical Support: Research Products: Clinical Products: Customer Service: Fax: Europe/International* Technical Support: Customer Service: europe@ebioscience.com Fax: Bender MedSystems GmbH Campus Vienna Biocenter Vienna, Austria * Customers outside North America and Europe may contact their ebioscience distributor listed on our website at

2 TABLE OF CONTENTS 1 Reagents Provided 3 2 Intended Use 3 3 Summary 4 4 Storage Instructions Simplex Kit 5 5 Specimen Collection 5 6 Preparation of Reagents and Samples 6 7 Representative Standard Curve 7 8 Performance Characteristics 8 9 Ordering Information (02)

3 3 This human Caspase-3 Simplex Kit must be used in combination with FlowCytomix human Basic Kit BMS8420FF. For test procedure, measurement and calculation of results please refer to FlowCytomix human Basic Kit BMS8420FF manual. 1 REAGENTS PROVIDED 1 vial (175 µl) Fluorescent Beads (20x) coated with monoclonal antibody to human Caspase-3, Bead Population B9 2 vials human Caspase-3 Standard (lyophilized): 1.2 µg/ml upon reconstitution 1 vial (350 µl) Biotin-Conjugate (20x) anti-human Caspase-3 polyclonal antibody 1 bottle (15 ml) Lysis Buffer Concentrate (10x) 2 INTENDED USE BMS82012FF is a bead based Analyte Detection System for quantitative detection of human Caspase-3 by Flow Cytometry. BMS82012FF is for research use only. Not for use in diagnostic or therapeutic procedures.

4 4 3 SUMMARY Caspases are a family of aspartate-specific cysteine proteases that act in a step-wise signaling manner like kinases. Caspases are present in all cells, recruitment of these proteases to oligomerized receptors leads to activation accompanied by autoproteolytic cleavage. Active caspases can proteolyze additional caspases generating a caspase cascade that cleaves proteins critical for cell survival. The final outcome of this signaling pathway is a form of controlled cell death termed apoptosis. The subgroup of caspases involved in apoptosis is called initiators or effectors. Caspase-3 cleaves substrate at the carboxyl terminus of aspartate residues. Active caspase-3 has two active sites and consists of two identical large (~ 20 kda) and two identical small (~ 10 kda) subunits that are derived from two precursor caspase-3 polypeptides. Caspase-3 is proteolytically activated by other caspases. Both subunits contribute to substrate binding and catalysis. The active site cysteine that covalently binds the substrate is located near the C-terminus of the large subunit. Active caspase-3 has two-fold symmetry, two active site pockets each residing on an opposite side. Caspase-3, together with caspases 8 and 9, is situated at pivotal junctions in apoptotic pathways. Caspase-3 appears to amplify caspase 8 and 9 initiation signals into complete commitment to apoptotic disassembly. For literature update refer to

5 4 STORAGE INSTRUCTIONS SIMPLEX KIT 5 Store kit and components at 2 to 8 C. The expiry of the kit components can only be guaranteed if the components are stored properly, and if, in case of repeated use of one component, the reagent is not contaminated by the first handling. 5 SPECIMEN COLLECTION Cell lysate samples were tested with this assay. Other biological samples might be suitable for use in the assay. Pay attention to a possible Hook Effect due to high sample concentrations (see chapter 8.4). Samples containing a visible precipitate must be clarified prior to use in the assay. Do not use grossly hemolyzed or lipemic specimens.

6 6 PREPARATION OF REAGENTS AND SAMPLES 6.1 Lysis Buffer 6 Pour the entire contents (15 ml) of the Lysis Buffer Concentrate (10x) into a clean 150 ml graduated cylinder. Bring to final volume of 150 ml with distilled or deionized water and mix gently. Store at room temperature. The Lysis Buffer (1x) is stable for 30 days. 6.2 Sample Preparation Cell Lysate Protocol Numerous extraction protocols can be used. The following protocol is provided as an example of a suitable extraction procedure, but should not be construed as necessarily being the method of choice. Users may wish to experiment with extraction procedures that work best in their hands. For suspension cells: Pellet by centrifugation, remove supernatant and proceed to Addition of Lysis Buffer. For attached cells: Remove supernatant from cells, wash cells once with PBS, harvest cells by scraping and gentle centrifugation, aspirate PBS, leaving an intact cell pellet (at this point the cell pellet can be frozen at - 80 C and lysed at a later date) and proceed to Addition of Lysis Buffer. Addition of Lysis Buffer: Resuspend the pellet in Lysis Buffer (1x) (we recommend a concentration of 5 x10 6 cells/ml.), incubate 60 minutes at room temperature with gentle shaking and transfer extracts to microcentrifuge tubes and centrifuge at 1000 x g for 15 minutes. Aliquot the cleared lysate to clean microfuge tubes and continue the test procedure (Alternatively lysates can be stored at -80 C and assayed at a later time. Divide lysates into small aliquots to avoid multiple freeze/thaw cycles.).

7 7 REPRESENTATIVE STANDARD CURVE 7 Table 1 Representative standard curve. Do not use this curve to derive test results. A standard curve must be run for each group of samples assayed. Concentration (pg/ml) Fluorescent Intensity (FI)

8 8 PERFORMANCE CHARACTERISTICS 8 Assay performance data presented in this manual was generated in house, and is considered typical for a routine experiment in our laboratories. Each laboratory using this product should establish its own performance characteristics, and these may vary from those presented in the manual. 8.1 Sensitivity The limit of detection of human Caspase-3 defined as the concentration resulting in a fluorescent intensity significantly higher than that of the dilution medium (mean + 2 standard deviations) was determined to be 20 pg/ml. The value shown depends on the type of flow cytometer used for analysis as well as on the respective instrument setup. The value shown is for guidance only. Optimum results for each machine can be achieved by following the instrument set up process. 8.2 Reproducibility Intra-assay Reproducibility within the assay was evaluated in 3 independent experiments. Each assay was carried out with 6 replicates of 4 lysate samples containing different concentrations of human Caspase-3 (high, medium high, medium low and low concentration). 2 standard curves were run on each plate. Data below show the mean intra-assay coefficient of variation for human Caspase-3 (see Table 2). It has been calculated to be 5.7%. Individual user data may vary due to differences in protein content of lysate samples. Table 2 The coefficient of variation of the human Caspase-3 concentration calculated for each sample. Sample 1 high (%) Sample 2 medium high (%) Sample 3 medium low (%) Sample 4 low (%) Mean intraassay (%) h Caspase

9 Inter-assay Assay to assay reproducibility within one laboratory was evaluated in 3 independent experiments. Each assay was carried out with 6 replicates of 4 lysate samples containing different concentrations of human Caspase-3 (high, medium high, medium low and low concentration). 2 standard curves were run on each plate. Data below (see Table 3) show the mean inter-assay coefficient of variation for human Caspase-3, calculated on 12 determinations of each sample. It has been calculated to be 6.9%. Individual user data may vary due to differences in protein content of lysate samples. Table 3 The coefficient of variation of the human Caspase-3 concentration calculated for each sample. Sample 1 high (%) Sample 2 medium high (%) Sample 3 medium low (%) Sample 4 low (%) Mean interassay (%) h Caspase Specificity Cross reactivity was tested with combinable analytes of Simplex and Multiplex Assays. There was no detectable cross reactivity observed. (For detailed information refer to Combination Table on Hook Effect Samples with expected concentrations two fold higher than the concentration of highest standard should be diluted 10 fold in Assay Buffer (1x) before assay performance to prevent false negative results due to a possible Hook Effect.

10 10 9 ORDERING INFORMATION North America Technical Support: Research Products: Clinical Products: Customer Service: info@ebioscience.com Fax: Europe/International* Technical Support: Customer Service: europe@ebioscience.com Fax: Bender MedSystems GmbH Campus Vienna Biocenter Vienna, Austria * Customers outside North America and Europe may contact their ebioscience distributor listed on our website at