I am attempting to stimulate isolated CD45+ cells with a cytokine stimulation cocktail before staining (for various subtypes of T cells) and then analyzing said cells by flow cytometry. Ideally, I would have around 90% viability, but I am getting around 50%...
I am stimulating the cells with the proper concentration of cocktail (https://www.biolegend.com/ja-jp/products/cell-activation-cocktail-without-brefeldin-a-9402?GroupID=BLG2181) for 5 hours (stimulation for T lymphocytes is recommended for 5 hours by the following paper: Article Optimization of stimulation and staining conditions for intr...
) in a dish in a typical cell culture incubator. I am also blocking ER and Golgi transport with Brefeldin A and Monensin. I tried doing the stimulation for only 3 hours, but viability wasn't improved.I may try stimulating my single cell suspension (derived from a single mouse liver and/or tumor) BEFORE isolating the CD45+ cells, but I'm afraid there will be too many cells for the stimulation to be effective. I could also potentially add the blocking agents later, but I'm afraid I will lose a significant portion of cytokine capture...
Any suggestions?
Hi Hannah,
What is your initial viability after purification (how do you purify your cells?) Do you work with fresh or frozen material?
What is the viability without Brefeldin A and Monensin?
Hello! Thanks so much for taking the time to consider my question.
I work with fresh material, dissociated into a single cell suspension. I haven't tested for viability at this point in the protocol.
Without Brefeldin A and Monensin, the viability is much better, around 80% (although it depends on the type of tissue). This viability value was determined after the cells were cultured in media for 5 hours.
Hi Hannah
"fresh material, dissociated into a single cell suspension"
Can you define the term "dissociated"? Do you use enzymatic digestion? This could be a critical point in your protocol for cell viability. For example, some lots of collagenase are very damaging to cells due to their trypsin-like activity.
Yes! I use Miltenyi Biotec's Liver and Tumor Dissociation Kits, which I believe are more on the gentler side in terms of enzymatic digestion. There is physical dissociation as well (chopping and rotating during enzymatic digestion using Miltenyi's "C-tubes").
It seems that your CD45s don't like to stay even 3 hours in your medium.
Bad 1640? (for example with low glucose or without glutamine (glutamax)).
Try to compare with any reference medium you can find by knocking on the doors of neighboring labs: e.g. different lot of RPMI, or HPLM or Iscove
Try addition of 1% BSA during the incubation period.
I do have GlutaMAX! It was suggested to me by another co-worker as well. Sounds like a good idea. I will consider trying BSA as well. Thanks so much!
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