S-adenosylhomocysteine FCM kit with Alexa Fluor® 488

1. Prepare the cells to be tested in the amount of 5 - 10 x 105/sample in Eppendorf tubes.

2. Centrifuge the cell suspension at 1500 rpm for 5 min, and discard the supernatant.

3. Fix the cells with 100 µI fixation buffer (either nucleus or cytoplasm system) for 30 min at room temperature in dark. For nucleus fixation, mix one portion of Nucleus Fix 1 with 3 portions of Nucleus Fix before use.

4. Centrifuge at 1500 rpm and remove the supernatant.

5. Prepare the proper amount (3 folds of the fixation buffer) of the working permeab由zation buffer by diluting Permeabilization Buffer stock solution 10 times with distilled water. Wash the samples with 100 µI permeabilization buffer.

6. Incubate with 100 µI permeabilization buffer for about 30min at room temperature, centrifuge and discard the supernatant.

7. Re-suspend with 100 µI permeablization buffer, followed by adding 10 µI 1 :5 diluted AF488-anti-SAH antibody included in the kit. Incubate at 4 °C for about 60 min.

8. Wash each sample with 1 ml PBS 2-3 times and re-suspend it with 0.5 ml PBS.

9. Run each sample through a Flow Cytometer.

Purpose : This kit is designed to measure the amount of SAH in the cytoplasm and nucleus compartments of cells conveniently and quickly using Flow Cytometry (FCM). 
About  the assay : S-adenosylhomocysteine, a sulfur一containing molecule, is an important molecule in methylation and transsulfuration. SAH does not have a distinguished absorption at 258-260 nm, accurate and timely determination of its concentration in various biological fluids and tissues has always been a challenging task. Therefore, finding a way that can quickly and specifically determine the amount of SAH becomes essential. With the advent of specific anti-SAH monoclonal antibodies, measure SAH from living cells directly, quickly and sensitively becomes possible with flow cytometry. Alexa Fluor® 488-conjugated mouse anti-SAH antibody has been used (Refer to Cat# MAF00302) after cells are fixed and permeabilized. In this kit, both cytoplasm and nucleus fixation/permeabilization buffers have been include. With nucleus fixation/permeabilization system, all targets from cytoplasm and nucleus will be detected, whereas with cytoplasm fixation/permeabilization buffer, only cytoplasmic portion of the target is detected.

 

1. Depending on the type of cells, the amounts and incubation time of cytoplasm and nucleus fixation and permeabilization buffers to be used might be slightly different.

2. Different cell cycles, metabolic status and health of cells may cause SAH level to change significantly in cells. Therefore, the amount of the antibody to use could be optimized for better results.