I am doing a pilot study to assess the cytotoxic abilities of different T lymphocyte populations when cocultured with B16 Ova In Vitro. I have seen different articles labeling their effector/cytotoxic T Cells with a target dye to gate them out and look at Annexin and PI positive cells. But I do not have any target dye to label my cells. Instead can harvest my cocultured tumor + T cells and first do a CD4 CD8 surface staining with antibodies and then follow Annexin PI staining protocol? I understand the 2 stainings are different but will that affect my apoptosis results in any way? Has anyone done this before and has had luck with it? Please help me to understand better. Thanks
Amended on 2 June 2019
I have searched for Annexin and Actin in my human protein database in vain (please see files; HepG2 Fucoidan and JMBT Alopecia). Interestingly, Actin-related proteins of NEDD9/Enhancer of filamentation 1/Cas scaffolding protein family member 2/Neural precursor cell expressed developmentally down-regulated protein 9/Renal carcinoma antigen NY-REN-12, ALKBH4/Alpha-ketoglutarate-dependent dioxygenase alkB homolog 4, NAA80/N-alpha-acetyltransferase 80/N-acetyltransferase 6, and SETD3/Actin-histidine N-methyltransferase are also absent in Humans.
Serum of 1y girl (with common cold; non-cancer) and healed hepatocyte HepG2 (cultured with fucoidan) have MICAL-like protein 2/Junctional Rab13-binding protein/Molecule interacting with CasL-like 2 at 4.1 μg/mg of serum protein and 1.1 μg/mg of cell protein, respectively.
Human breast milk (from healthy mother) only has Actin-like protein 7A at 2.2 μg/mg of milk protein.
MICAL-like protein 2 and Actin-like protein 7A seem to be important in membrane vesicle transport in the normal cells with no link to Actin, since Humans has no Actin. Therefore, I have now recognized that Fucoidan effect on Cancer is due to increase of Membrane Components (please see file again; Hep G2 Fucoidan).
I am grateful to Dr. Achintyan Gangadharan (Department of Immunology, University of Texas MD Anderson Cancer Center, Houston, TX, USA) for leading me to this important finding.
Therefore, notorious phenomena of Exocytosis, Exosome, Apoptosis, Telomere, Programmed cell death and Autophagosome are absent in the Humans. Therefore, notorious Annexin V/Propidium Iodide (PI) assay cannot quantitatively determine Apoptosis in humans at all. Annexin V-FITC, Annexin V-7-AAD (7-Amino-Actinomycin D) and Annexin V-DAPI (4',6-diamidino-2-phenylindole) methods are also not applicable to humans. Further, Flow cytometry is not quantitative due to violence against Beer-Lambert law. Immunological methods (such as ELISA) are also not quantitative due to non-specific binding reactions by Immunoglobulin G/IgG made by either monoclone or multiclones.
Nematode of Caenorhabditis elegans and anaerobic parasitic Amoebozoan of Entamoeba histolytica may have Actin. Annexin may be present in Adrenal glands of Bovine and marbled electric ray (Torpedo marmorata).
This large Species differences clearly indicates that "Theory of Evolution" is completely false.
Further, I have searched for CD8. CD8a and CD8b are present in Humans. However, CD4 is not present in Humans. CD4 may be present in Mouse, and the representative Species differences is occurring in T cell differentiation.
As a reference, Humans has following CDs.
CD1d, CD5 antigen-like/IgM-associated peptide, CD8-alpha, CD8-beta, CD13, CD14, CD20 antigen-like 7/Membrane-spanning 4-domains subfamily A member 10, CD21, CD29, CD30, CD40, CD44, CD45, CD49b, CD49d, CD51, CD55/Complement decay-accelerating factor, CD56, CD58, CD61, CD69, CD88, CD98, CD99, CD103/Integrin alpha-E/HML-1 antigen, CD104, CD107a, CD116/Granulocyte-macrophage colony-stimulating factor receptor alpha chain/GM-CSF-R, CD117, CD127, CD129, CD152, CD154, CD163, CD206/Macrophage mannose receptor 1, CD233/Anion exchange protein 1/Band 3 anion transport proteinSLC4A1, CD243, CD292, CD331, CD332, CD340.
By the way, even reliable data obtained by HPLC-photometric determination methods should be handled with by non-parametric statistics since nature does not have Normal distribution (please see file; Dr. Terentyeva Urine BIN).
I'm confused. If you do CD4/8 immunostaining, gate that population, then look for PI/annexin subpopulations, I'm not sure what the problem is. As long as they are labeled with separable fluors and compensated properly, you've answered your own question.
I have the same question, recently I saw and article that does exactly that, staining with CD4/CD8 and Annexin V, however they use biolend Annexin and I have Thermo fisher, so I'm going to make a test using PBMCs first. I'll let you know my results. Which brand of Annexin are you using?
In theory you can stain with anti CD4/CD8 after the AV staining, like a normal surface staining. That shouldn't alter your results. I agree with Matt, if you select your CD4/CD8 gate and then analyze over your negative population, that should be your target cells.
Other thing you can do is use CD45.1 effector cells and CD45.2 target cells for example.
Eduardo Duran does it have to be in that order? do you have a protocol or recommendations regarding this? thank you
Hi Sarah Ratkovich-Gonzalez , I remember that you shoul perform the surface staining first and then stain with AV (and 7AAD for example). That is because for AV staining you need to you a "special" binding buffer.
Eduardo Duran Thank you so much! you save me a lot of time!
- Badges
- Science topic
- Similar topics
- Biological Science
- Immunology