Annexin V Apoptosis Plate Assay KitAD12 Product Manual

　Apoptosis is a type of programmed cell death that plays an important role in maintaining homeostasis and developmental processes in plants and animals. For example, any abnormal cells during cytogenesis are eliminated by apoptosis. In the early stage of apoptosis, the phosphatidylserine (PS) present in the inner cell membrane migrates to the outer cell membrane. This specific change allows the identification of apoptotic cells, because Annexin V binds specifically to PS in the presence of calcium ions. Hence, by using fluorescently labeled Annexin V, apoptotic cells can be detected. Generally, the apoptotic cells are detected by flow cytometry or fluorescence microscopy. However, these methods require time to process multiple samples.
　This kit includes a reagent (Quenching Buffer) that eliminates the fluorescence of any extracellular fluorescently labeled Annexin V that is not bound to PS, so it is possible to rapidly measure multiple samples using a plate reader without a washing process.

Annexin V - FITC	× 1
Quenching Buffer	11 ml × 1

Store at 0–5 °C

• Fluorescence microplate reader
• 96-well black microplate (clear bottom)
• 20–200 μl multichannel pipette
• 100–1000 μl、20–200 μl、2–20 μl micropipettes
• Conical tube
• Microtube

• Equilibrate reagents to room temperature prior to use.
• Briefly centrifuge the tubes before opening to ensure the content is at the bottom.
• Analysis of samples in triplicate is recommended for accuracy.

Preparation of Annexin V - FITC stock solution

Add 250 µl of double-distilled H₂O (ddH₂O) to the Annexin V - FITC tube and dissolve by gently pipetting.

• The Annexin V - FITC stock solution is stable for 1 month when stored at 0–5 °C.

Preparation of working solution

Add Annexin V - FITC stock solution and Quenching Buffer to a conical tube and mix by gently inverting the contents to obtain a working solution.

• Please refer to the Table for the amounts to be mixed.
• Please prepare enough working solution for samples and blank (n=3).
• When using a multichannel pipette, please prepare the working solution for sample wells, blank wells and two more wells.
• Prepare the solution just before use and protect it from light. Please use up the working solution within that day.

Examples of working solution preparation

	for 48 wells	for 96 wells
Annexin V - FITC	125 μl	250 μl
Quenching Buffer	3000 μl	6000 μl

1. Seed cells in a 96-well black microplate (clear bottom, 1–5×10⁴ cells/well), and culture overnight in an incubator (37 °C, 5% CO₂).
2. Stimulate the cells with drug as needed (add drug solution to wells). 
   Note: Please make sure that the culture supernatant volume is 100 µl.
3. Add 60 µl of working solution to each well.
   Note: For the blank, add a working solution to medium-only wells.
4. Incubate the microplate at room temperature for 15 minutes.
   Note: Please protect the microplate from light.
5. Measure the fluorescence intensity using a fluorescence microplate reader (bottom reading, Ex/Em = 488 nm/525 nm).
6. ​To obtain the experimental reading, subtract the measured value of the blank from the value measured for each sample.

Evaluation of induction of apoptosis in HeLa cells

1. HeLa cells were seeded in a 96-well black microplate (clear bottom, 2×10⁴ cells/well, in MEM containing 10% fetal bovine serum and 1% penicillin-streptomycin) and cultured overnight in an incubator (37 °C, 5% CO₂).
2. The medium was removed from three wells per timepoint (6, 4, and 2 hours before the addition of working solution), and 5 µmol/l staurosporine solution (100 µl, in MEM containing 10% fetal bovine serum) was added to the cells (Figure 1, 2). The cells were incubated at 37 °C in 5% CO₂.
   Note: For the blank, 100 µl of MEM only was added to wells.
   Note: For the control, 100 µl of MEM without staurosporine was added to cells immediately before the addition of the working solution.
   Note: Staurosporine treatment was performed to align the timing of adding the working solution in the next step 3.
3. A working solution (60 µl) was added to each well, and the microplate was incubated at room temperature for 15 minutes.
   Note: The microplate was protected from light.
4. The fluorescence intensity was measured using a microplate reader (Infinite M200 PRO, Tecan Trading AG, bottom reading).
5. The result for the fluorescence intensity was calculated by subtracting the blank value from the value measured for each sample (Figure 3).

Figure 1.　Plate format

Figure 2.　Staurosporine treatment procedure

Figure 3.　Changes in fluorescence intensity with staurosporine treatment time (Ex/Em = 488/525 nm)