Hi I was wondering if anyone can shed some light on this problem I'm stuck with. (Please check the attached PDF file)
[Background]
I isolated naive T cell (or what I thought to be) using a negative selection kit, so that the cells are untouched.
I used unstained, pre-isolation samples (total splenocytes) as negative control to set up a gating logic.
Unfortunately I have confirmed using flow cytometry that the isolated population contains CD44+ T cells. (Effector Memory T cells? [TEM])
Since the kit that I used contains anti-CD44 biotin antibody, it should have removed the TEM from the population.
[Question]
After inquiring the company where I purchased the kit from, they told me that I have to use the stained, pre-isolation sample as negative control.
They've also told me that by their specific gating logic, the population is TEM-free and ready to use but I still don't quite understand the logic behind the negative control and the gating logic they have used.
Can someone please help me understand my problem?
Thanks in advance.
Heesue
A stained, pre-isolation sample should be your negative control and more appropriate compared to an unstained control. It would show the distribution of CD4+ populations in your sample. After depletion, only negatively selected population should remain.
Hope this helps.
As Jesvin suggested, you should include pre-isolation sample to check the distribution of CD4+ naive vs. memory cells in your sample. Only comparison of those 2 samples will allow you to see where CD44hi cells are supposed to be on your plots. In my experience, they are clearly distinguishable using CD44 staining on spleen CD4+ cells from healthy mouse. You might consider adding CD62L staining (naive CD4+ cells should be CD62L+, memory CD4+ cells should be CD62L-), but be aware that CD62L is easily shedded from the cell surface and might not be the best choice. Other option would be to use CD45RB (naive cells should be CD45RB+, memory cells CD45RB-). Whichever marker you choose it will still be important to compare sample before and after isolation.
However, I have few questions of my own - how are you detecting CD44hi cells after purification? Are you staining with CD44 labeled Ab that is of different clone than one in the kit? Moreover, how does CD44 Ab bind selectively to memory but not naive cells (as they are are all CD44+ (some low, some hi)) for separation?
By the way, I noticed that your gates are not ideal. I would recommend excluding smaller population at ~50K on FSC x-axis. According to my experience, these are dead/dying cells that are better to exclude from analysis.
@Berislav
Thanks for answering my question!
I haven't realised that the anti-CD44 antibodies were from the same clone. Thanks for pointing out; I wouldn't have realised it if you didn't pointed it out for me.
You are absolutely right about the CD44-/+ matter. It's a bad habit of mine to call CD( )low as CD( )negative. Thanks again for the correction.
Thank you Jesvin and Vamsi for answering my question and giving me great advice.
I'm new to this field and your advises definitely clarified things for me :)
- Badges
- Science method