Multiplex Assay Kit for Caspase 9 (CASP9) ,etc. by FLIA (Flow Luminescence Immunoassay)

APAF3; ICE-LAP6; MCH6; Apoptosis-Related Cysteine Peptidase; Cysteinyl Aspartate Specific Proteinases 9; Apoptotic protease-activating factor 3; ICE-like apoptotic protease 6

(Note: Up to 8-plex in one testing reaction)

Specificity

This assay has high sensitivity and excellent specificity for detection of Caspase 9 (CASP9) ,etc. by FLIA (Flow Luminescence Immunoassay).
No significant cross-reactivity or interference between Caspase 9 (CASP9) ,etc. by FLIA (Flow Luminescence Immunoassay) and analogues was observed.

Recovery

Matrices listed below were spiked with certain level of recombinant Caspase 9 (CASP9) ,etc. by FLIA (Flow Luminescence Immunoassay) and the recovery rates were calculated by comparing the measured value to the expected amount of Caspase 9 (CASP9) ,etc. by FLIA (Flow Luminescence Immunoassay) in samples.

Matrix Recovery range (%) Average(%)
serum(n=5) 98-105 102
EDTA plasma(n=5) 80-98 86
heparin plasma(n=5) 78-97 82
sodium citrate plasma(n=5) 98-105 102

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level Caspase 9 (CASP9) ,etc. by FLIA (Flow Luminescence Immunoassay) were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level Caspase 9 (CASP9) ,etc. by FLIA (Flow Luminescence Immunoassay) were tested on 3 different plates, 8 replicates in each plate.
CV(%) = SD/meanX100
Intra-Assay: CV<10%
Inter-Assay: CV<12%

Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Caspase 9 (CASP9) ,etc. by FLIA (Flow Luminescence Immunoassay) and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample 1:2 1:4 1:8 1:16
serum(n=5) 78-101% 84-103% 87-95% 95-102%
EDTA plasma(n=5) 79-96% 92-99% 91-101% 89-98%
heparin plasma(n=5) 80-91% 96-104% 97-105% 99-105%
sodium citrate plasma(n=5) 80-89% 90-97% 90-97% 80-96%

Stability

The stability of kit is determined by the loss rate of activity. The loss rate of this kit is less than 5% within the expiration date under appropriate storage condition.
To minimize extra influence on the performance, operation procedures and lab conditions, especially room temperature, air humidity, incubator temperature should be strictly controlled. It is also strongly suggested that the whole assay is performed by the same operator from the beginning to the end.

Reagents and materials provided

Reagents Quantity Reagents Quantity
96-well plate 1 Plate sealer for 96 wells 4
Pre-Mixed Standard 2 Standard Diluent 1×20mL
Pre-Mixed Magnetic beads (22#:CASP9) 1 Analysis buffer 1×20mL
Pre-Mixed Detection Reagent A 1×120μL Assay Diluent A 1×12mL
Detection Reagent B (PE-SA) 1×120μL Assay Diluent B 1×12mL
Sheath Fluid 1×10mL Wash Buffer (30 × concentrate) 1×20mL
Instruction manual 1

Assay procedure summary

1. Preparation of standards, reagents and samples before the experiment;
2. Add 100μL standard or sample to each well,
    add 10μL magnetic beads, and incubate 90min at 37°C on shaker;
3. Remove liquid on magnetic frame, add 100μL prepared Detection Reagent A. Incubate 60min at 37°C on shaker;
4. Wash plate on magnetic frame for three times;
5. Add 100μL prepared Detection Reagent B, and incubate 30 min at 37°C on shaker;
6. Wash plate on magnetic frame for three times;
7. Add 100μL sheath solution, swirl for 2 minutes, read on the machine.

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Magazine Citations
Reprod Sci. Role of mitochondria in ciprofloxacin-induced apoptosis in murine sperm cells. Pubmed: 23420823
Molecular Neurobiology Ursodeoxycholic Acid Ameliorates Apoptotic Cascade in the Rotenone Model of Parkinson’s Disease: Modulation of Mitochondrial Perturbations Pubmed:25502462
BMC Cancer Saikosaponin d induces cell death through caspase-3-dependent, caspase-3-independent andmitochondrial pathways in mammalian hepatic stellate cells. pubmed:27461108
Exp Lung Res Stretch-induced apoptosis in rat alveolar epithelial cells is mediated by the intrinsic mitochondrial pathway. pubmed:28394655
The American Journal of Chinese Medicine Saikosaponin a Induces Apoptosis through Mitochondria-Dependent Pathway in Hepatic Stellate Cells pubmed:28231747
Frontiers in Pharmacology Docking Studies and Biological Evaluation of a Potential β-Secretase Inhibitor of 3-Hydroxyhericenone F from Hericium erinaceus. pubmed:28553224
Clinical And Experimental Pharmacology And Physiology  Diallyl sulfide alleviates cisplatin‐induced nephrotoxicity in rats via suppressing NF‐κB downstream inflammatory proteins and p53/Puma signalling pathway Pubmed:29266336
Environmental Toxicology Antihepatocarcinogenic activity of whey protein concentrate and lactoferrin in diethylnitrosamine‐treated male albino mice Pubmed: 31087429
European journal of medicinal chemistry Synthesis of novel S-linked dihydroartemisinin derivatives and evaluation of their anticancer activity Pubmed: 31216504
Odontology Effects of different pulp-capping materials on cell death signaling pathways of lipoteichoic acid-stimulated human dental pulp stem cells Pubmed: 33206337
Environ Sci Pollut Res Int Potentiation of the apoptotic signaling pathway in both the striatum and hippocampus and neurobehavioral impairment in rats exposed chronically to a low− dose of … Pubmed: 32915453
SAGE Journals Insights Into Protective Mechanisms of Dandelion Leaf Extract Against Cisplatin-Induced Nephrotoxicity in Rats: Role of Inhibitory Effect on Inflammatory and Apoptotic …
Eur J Pharmacol Potential cardioprotective effect of octreotide via NOXs mitigation, mitochondrial biogenesis and MAPK/Erk1/2/STAT3/NF-kβ pathway attenuation in isoproterenol … Pubmed:35500641
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