I'm doing a Mixed Lymphocyte Reaction and sorting out alloreactive T cells by FACS.
Briefly, I treat feeder cells with 25ug mitomycin C for 30 minutes and wash them 2x with RPMI+10% FBS to render them non-proliferative. I resuspend the feeder cells in AIM V media +10% heat inactivated human AB serum.
Next, I sort T cells from my responder population by MACS (Pan T cell isolation kit, Miltenyi Biotech). I spin down the sorted T cells, stain them in 5uM CFSE, wash them, and resuspend in AIM V media +10% heat inactivated human AB serum.
I plate 200k of each population in 200uL total volume on a 96 well plate (U bottom wells).
Prior to FACS, I stain the cells with CD3:APC-CY7, CD4:AF700, and CD8:BUV395. The goal is to sort out CD3+CD4+ and CD3+CD8+ alloreactive T cells for downstream analysis.
I have a lot of debris outside my scatter gates. Even under the PCM, my cells look awful. What am I doing wrong?
What is your IL-2 dose? Are you adding any peptide or anti-CD3 mAb? What clone and dose?
Hi Govinda,
I am not using any IL-2.
The MLR media is AIM V supplemented with 10mM HEPES and 10% heat inactivated human AB serum.
I use 2% PHA as a stimulant in my positive control groups. I do not presently use anti-CD3.
Effectively, I'm attempting to replicate an experiment done by Megan Sykes (PMCID: PMC4360892) using a very different patient population. The paper does not mention the use of IL-2 in culture. Is this something that I necessarily need to be doing?
Yeah, they don't seem to be using IL2. I suppose the assumption is that accessory cells in the media are sufficiently conditioning the media with IL-2. We always use IL-2 (300 U/mL) final when doing T-cell activation and expansion. If you're having trouble expanding any cells out though, it might be worth adding it in.
I'm guessing that they avoid adding IL-2 in the Sykes paper to avoid expanding Tregs along with the donor-reactive clones. If you are doing a similar sequencing study and want to avoid contaminating your reads with Tregs, you might consider including another gate to exclude CD25hi cells and capture CD25lo cells either before or after MLR
I'll also point out that it would be normal to see a lot of dead cells/dying cells/debris in FCM as the MMC-treated stimulator cells will be dropping out of culture around day 6
Hello
i agree with Govinda Sharma .
Look at link here will help you
Good Luck
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2327014/
CFSE can be very toxic. Be cautious when using CFSE labeling cells and 80% cells will be apoptotic. Try to adjust the concentration of CFSE and duration of staining if you can.
Certainly, you can use PMA and Iono to test your cells whether they are viable as a positive control.
CFSE is quite toxic. I used to stain cells with CFSE in PBS and had the same problem. The latest recommended protocol for CFSE staining involves staining the cells in complete media (ie making up the staining solution in complete media), staining for 5mins then washing with complete media. My experience is that using this method you can add very high concentrations of CFSE with minimal toxicity and very bright staining. This paper is by the people who did all the work on CFSE and explains.
1. Quah, B. J. C. & Parish, C. R. New and improved methods for measuring lymphocyte proliferation in vitro and in vivo using CFSE-like fluorescent dyes. J. Immunol. Methods 379, 1–14 (2012).
Article New and improved methods for measuring lymphocyte proliferat...
Hi, Some amount of cell loss has to be anticipated as T cells die on activation itself. CFSE toxicity could be one of the reasons you see lot of death as mentioned above. However, complete Media or PBS containing serum is not a good idea as CFSE is known to bind free amines in aqueous solutions. Titrating the concentration according to the cell concentration helps to overcome this. Alternatively a less toxic and equally efficient option is VPD450, a violet laser excitable dye at 450nm. Its minimal spill over to FITC channel makes it ideal to use with GFP positive cells.