I am looking for suitable positive control for early apoptosis detection by using Annexin-V in FACS.
if anyone has an experience with such drug kindly assist me in regards of drug concentration to be used and total time of treatment to induce early apoptosis in B cells.
Thanks in advance
Check the transition of LC3alpha to LC3betta; use fluorescent microscopy and/or Western blot. Drug concentration will be different because of different response of autophagy reaching several micrograms/ml.
Look the article of Iradyan et al. Targeting Degradation of EGFR through the Allosteric Site Leads to Cancer Cell Detachment-Promoted Death. Cancers, 2019 here: Article Targeting Degradation of EGFR through the Allosteric Site Le...
Do you need a positive control for your Annexin V staining? In this case, you can simply expose a sample of your cells of interest to high temperature (ie. 55C for about 20 min) or UV radiation (under your BSL hood with UV turned on) and then mix untreated cells with treated ones (so that your tube will contain both a positive and a negative population).
Annexin V does not induce apoptosis by itself when used a FACS staining, but if you are asking how to use it... stain your cells as you always do, wash them and resuspend them in Annexin V binding buffer (ie. saline with HEPES and divalent cations such as Ca, commercially available) and add 3-5 uL/100uL of fluorophore-conjugated Annexin V. Analyze your cells at FACS (w/o wash).
Here is a protocol for Fas antibody mediated apoptosis induction.
https://www.abcam.com/protocols/induction-of-apoptosis-in-cells
good luck.
Actinomycin D works well too.
https://www.pnas.org/content/113/38/10666
I used 0.5–5.0 µM doxorubicin as a positive control in my apoptosis studies (6-48 h). You can find the method in the following publication:Article Comparison of Cellular Death Pathways after mTHPC-mediated P...
With the addition of propidium iodide (PI) into your staining procedure, you will be able to discriminate between early and late stage apoptosis with the annexin V/PI assay. PI will stain DNA in addition to annexin V-stained phosphatidylserine, which is characteristic for apoptosis at a later stage. Early apoptotic cells remain PI-negative.
I used cisplatin (10µM) as positive control using Annexin V-Cy3TM apoptosis detection kit assay.
Keep in mind that using anthracycline or actinomycin as inducer of apoptosis may result in staining of your cells (these chemotherapeutic drugs are fluorescent). For example, Daunorubicin or Doxorubicin are excited by the blue laser and emit with a maximum around 600-625 nm and, as they are cell permeant and bind DNA, their fluorescence does not correlate with cell death.
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