Does anyone know the exact effects of irradiation of PBMCs (publication??) and if it is possible to freeze irradiated PBMCs and thaw them when needed. Also what radiation dose is recommended? Thank you!
See the link below. In general, freezing irradiated PBMCs is not advised. One can prepare extracts and freeze them.
Link: http://www.radioprotection.org/articles/radiopro/pdf/2008/05/000161.pdf
Peripheral ly are generally rradiosensitve cells and upon irradiation die by apoptosis. So the doses and time will depend on your needs, what do you want to achieve?
http://www.ncf-net.org/radiation/VokurkovaRadiosensitivityOfCD56brightNKcells.pdf
actavet.vfu.cz/media/pdf/avb_2011080010113.pdf
http://www.actabp.pl/pdf/2_2008/381.pdf
http://www.ncf-net.org/radiation/VokurkovaRadiosensitivityOfCD56brightNKcells.pdf
Storing pbmc's is not recmonded as they are primary cells and are very sensitive to stress. Most experiments on PMBC's are performed with in 2-3 sab culture. However You can preseve they lysate from pbmc at -80°C for a month or Two.
Radiation dose and dose rate depends on the types of study you wish to perform. for most studies LD90, LD50 and LD10 is prefered which varies from cell to cell. You can perform kinetic blue and MTT assay in order to find out these doses and choose youself the dose you wish to perform your studies on
Hello Martina, hello Sumit,
thank you for your answer! I want to use the irradiated PBMCs for HIV virus culture. therefore I want to understand the advantage of using irradiated PBMCs in contrast to un-irradiated PBMCs. So far, I understand that irradiated PBMCs in combination with PHA stimulated PBMC activate resting CD4+ cells. I found a book recommending 5000R. Do you have any experience in this method?
http://www3.unisi.it/fisica/dip/pers/bottigli/EXCALIBUR/Pubblicazioni/ncc9281.pdf
Effect of cryo-preservation on the response of different biological systems to γ-ray exposure: A feasibility study
please also read this article
Dear Seda,
unfortunately I am no expert in HIV cultures. Genetally, although irradiation by 50 Gy will kill the PBMC, it will also trigger changes in their cytokine secretion and may alter dendritic cells performace, thus enhancing T-cell proliferation in co-cultured noniradiated cells (but it is a bit controversial topic, see eg.
http://onlinelibrary.wiley.com/doi/10.1016/j.cellbi.2003.12.006/pdf
5000R or 50Gy is alot of radiation on i would suggest you to cross check that one..there would be nothing left of PBMC after absorbing that much ammount of radiation. As far as PHA is considered its really importatnt for pbmc to proliferate. The doubling time that we were getting was arnd 96hrs.. U need to standerdize all of it nd even if u wish to perform ur studies at sensetizing dose you need to know radiation LD50 for your cells. I'd suggest you choose for slower dose rates as you want them to grow and proliferate.
Using the PBMCs as "feeder cells" 50 Gy OR 5000 rad is sufficient, Seda. I typically irradiate my feeder cells with 50 Gy, (if you can let them rest overnight) remember to wash them and resuspend them in the appropriate buffer/media before addition to your HIV-specific T-cell cultures. I'm presuming you are trying to expand these HIV-specific T-cells and have a monoclonal or polyclonal T cell line?
Hey Craig,
thank you for your answer! So you let the irradiated cells rest before you feed them to your cells? What kind of medium do you use? I use RPMI with 10% FBS, 1% P/S, PHA and IL-2 at the day of irradiation. But I also feed them right after the irradiation.I want to cultivate the virus infected cells, to have a detectable virus-titer (p24-analysis). It didn't work so far. I don't know if it's the fact, that these patients are on ART or that my protocol needs to be changed. Would be happy to hear from you!
I find I need to rest irradiated cells overnight in RPMI-1640 10% FBS, 1% P/S. If i add them straight away I get no expansion, even with non-specific stimulants like CD3/CD28. After allowing them to rest (at 37'C with 5% CO2) I wash and resuspend in RPMI with 10% FBS, 1% P/S, IL-2 and the stimulant (i.e. CD3/CD28). I don't think the patients being on ART with low viral loads should be a problem. If this still doesn't work I would look adding extra Glutamine (i.e.1mM of GLUTAMAX) (I presume your RPMI-1640 is Glutamine positive?) and maybe adding sodium pyruvate (1mM) and check the FBS is correctly heat-inactivated at 56'C for at least 1 hour and sterile filtered through a 0.22um PVDA membrane filter / syringe filters. Keep me updated with what you find, I would love to know what you find.
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