神奇紅Caspase-3/7 檢測試劑盒

使用 Magic Red Caspase 3/7 Assay Kit 實時檢測 caspase 3 和 7 的活性。Magic Red 試劑在被活性 caspase 3 和 7 酶切割後發出螢光。使用熒光顯微鏡、盤式螢光分析儀或流式細胞儀分析螢光訊號。

Background Caspases play important roles in apoptosis and inflammation. Detection of DEVDase enzyme activity (caspase 3 activity) is a reliable method for assessing apoptotic activity in experimental cell populations. The presence of the active form of caspase 3 & 7 in apoptotic cells causes the hydrolysis of the two DEVD target sequences from the Magic Red cresyl violet fluorophore, converting it to the fluorescent form. As apoptosis progresses and caspase activity increases, the red fluorescent signal increases. Magic Red excites at 590 nm and emits at 628 nm and can be analyzed using fluorescence microscopy, a fluorescent plate reader, or flow cytometry. Samples may also be analyzed with Acridine Orange dye or Hoechst stain to visualize lysosomal organelle structure or detect changes in nuclear morphology respectively.

Reagent Name MR-(DEVD)2

Target Caspase-3/7

Excitation / Emission 590 nm / 628 nm

Method of Analysis Fluorescence Microscope, Fluorescent Plate Reader, Flow Cytometer

Sample Type Cell culture, tissue

Storage 2-8°C

Country of Origin United States

Protocols

1. Prepare samples and controls

2. Reconstitute Magic Red with 100 or 400 µL DMSO

3. Dilute Magic Red 1:5 with diH2O

4. Add diluted Magic Red at 1:15-1:30 to suspension cells

5. Incubate while protected from light.

6. Watch color start to develop within 15 minutes of addition of Magic Red

7. If desired, label with additional stains, such as Hoechst, DAPI, or an antibody.

8. Analyze with a fluorescent microscope, fluorescent plate reader, or flow cytometer. Magic Red has a maximum excitation at 592 nm and emission at 628 nm. Good fluorescence images can be obtained at 510-560 nm excitation and >610 nm emission.

****If working with adherent cells, please see the manual for additional protocols.

Kit Contents

Kit# 935: 25 Tests

MR-(DEVD)2 Reagent, 1 25-test vial, #6131

Hoechst 33342 Stain, 1 mL, #639

Acridine Orange Stain, 1 mL, #6130

Kit Manual

Frequently Asked Questions

Question: The customer wish to combine in the same sample the Magic Red staining and Annexin V staining. The analysis of the samples will be performed on a flow cytometer. In the manual, page 10, section: single satin Flowcytometry, after the MR incubation there is no wash step. Since the Annexin V staining procedure can be performed only with a staining buffercontaining calcium, can the MR be performed in this buffer instead of the cells media? So that the customer will begin with the MR staining and in the last 15 min she will add the Annexin V. Another option can be perhaps to stain with the MR, wash and replace it to the Annexin V buffer for future 15 min staining. Will the MR “survive” a centrifuge? Answer: We have not evaluated our Magic Red substrates in combination with Annexin V, so unfortunately can’t say with certainty that this will work. However, that said, we would recommend against the proposed sequence where the customer stains with MR, washes, resuspends in Annexin V buffer, and then completes the Annexin V staining step. MR substrate is not covalently coupled in the cell and the signal would be washed away in the centrifugation step. The customer can try completing Annexin V staining and then carrying our MR staining afterwards in the Annexin V buffer. We would once again caution that the use of this technique will need to be evaluated experimentally. We recommend including positive and negative single stain controls for interpretation of the results.