5X Annexin V-FITC Apoptosis Detection Reagent (ab14082)

Hi there,

I’m interested in your product: 5X Annexin V-FITC Apoptosis Detection Reagent (ab14082)

1. According to your protocol for flow cytometry analysis, the concentration of the product is 0.25mg/ml, and we need to

add 1µl of Annexin V-FITC and 1µl Propidium Iodide into the cells suspension. May I know what is the actual concentration of the

1µl of Annexin V-FITC and 1µl of Propidium Iodide added into the cells suspension?

Many Thanks for your help.

ANSWER:

Thank you for your enquiry and your interest in our products.

The concentration of this product (ab14082) is 0.15 mg/ml, which is equivalent to 0.15 µg/ul. This is added to 500 µl makes it 0.15 µg/500 µl, which is 0.3 µg/ml. The PI is 1 mg/ml which is equivalent to 1 µg/µl, and in 500 µl it would make 2 µg/ml. (But note that the PI reagent actually has to be diluted to 250 µg/ml before use, which would mean that if this is done, the final concentration of PI in the 500 µl solution would be 0.5 µg/ml).

I hope this helps and if I can assist further, please do not hesitate to contact me.

Hi, i am having problems getting the annexin antibody to work despite using the annexin-v binding buffer. I mix the cell samples in a TALP media and dilute that 1:9 with binding buffer. I incubate the samples for 15 min in the dark. Do you have any suggestions and what is function of the binding buffer?

ANSWER:

Thank you for contacting us for technical support, I'm sorry to hear you are experiencing problems with ab14082, I am trying to find out from the lab the function of the binding buffer and as soon as I find out I will forward to you this information. Here is the recommended protocol with this antibody, I will ask my colleagues to add it to the datasheet:

A. Incubation of cells with Annexin V-FITC: 1. Induce apoptosis by desired methods. 2. Collect 1 x 105 cells by centrifugation. 3. Resuspend cells in 500 µl of 1X Annexin V Binding Buffer. 4. Add 1 µl of Annexin V-FITC and 1 µl Propidium Iodide. 5. Incubate at room temperature for 5 min in the dark. Proceed to B or C below depending on method of analysis.

B. Quantification by Flow Cytometry: Analyze cells by flow cytometry (Ex. = 488 nm; Em. = 530 nm) using FL1 channel for detecting Annexin V-FITC staining and FL2 channel for detecting PI staining. For adherent cells, trypsinize and gently wash cells with serum-containing medium before incubation with Annexin V-FITC.

C. Detection by Fluorescence Microscopy: 1. Place the cell suspension on a glass slide, and cover with a glass coverslip. For analyzing adherent cells, grow cells directly on a coverslip. Following incubation, invert coverslip on a glass slide and visualize cells. The cells can also be washed with 1X Annexin V Binding Buffer and fixed in 2% formaldehyde before visualization. (Cells must be incubated with Annexin V-FITC before fixation because any cell membrane disruption can cause nonspecific binding of annexin V to PS on the inner surface of the cell membrane.) 2. Observe the cells under a fluorescence microscope using a dual filter set for FITC and rhodamine, or separate filters. Cells that have bound Annexin V-FITC will show green staining on the plasma membrane. Cells that have lost membrane integrity will show red PI staining throughout the nuclei and a halo of green staining (FITC) on the plasma membrane.

I hope this protocol will help you and will forward to you the information about the binding buffer as so as I receive it, Thank you for your patience,

I would like to know the concentration of the Annexin V- FITC conjugate in your Annexin V- FITC Apoptosis Detection Reagent Kit (ab14082)

ANSWER:

Thank you for your enquiry. The associated concentration for the Annexin V- FITC conjugate in the Annexin V- FITC Apoptosis Detection Reagent Kit is 250 ug/ml. Please contact us again if you require further assistance.