I am new to Immunology and am mainly interested in studying total RNA from Naive CD4 T, Th1,Th2 and Th17 cells. I would prefer to directly FACS sort CD4 T cells and then stimulate them. but the beads Enrichment/separation step is a bit confusing for me. Any insight will be really Helpful.
I suppose both +ve and -ve selection methods can be used for CD4 T cell separation, But I see protocols using CD4+ T cell enrichment kits rather than doing direct FACS separation or using beads enrichment followed by FACS. Why is this enrichment step useful?
I am new to Immunology and am mainly interested in studying total RNA from Naive CD4 T, Th1,Th2 and Th17 cells. I would prefer to direcly FACS sort CD4 T cells and then stimulate them. but the beads Enrichment/separation step is a bit confusing for me. Any insight will be really Helpful.
For your polarisations, you would benefit from using naive T-cells. FACS sorting should always generate a higher purity (>95%, often >98%), but the cells will need to be stained with fluorescently conjugated antibodies (at least a viability dye and CD4 and probably CD45RA/RO). These antibodies can remain on the cell surface for a few days, and can theoretically interfere with downstream FACS analysis if you use the same fluorochrome for staining IFNg/IL-4/IL-17A in your Th-polarised cells. Sometimes when people sort a lot of cells, they do a column/bead enrichment for CD4+ T-cells, then use cell sorters to purify the naive T-cell subset. This speeds up the FACS process, as this can cost up to £60/hour in our facility here in Birmingham. However, there also exist many commercial kits from mainstream suppliers for isolating naive human CD4+ T-cells from PBMC. If you go this route, I suggest using the 'negative selection' kits, as these are designed to isolate cells through removing all cells that you are not interested in (e.g. B-cells, monocytes, CD8+ T-cells NK, memory T-cells, Treg), leaving behind your CD4+ T-cells 'untouched' by antibodies or magnetic particles.
Yes, the bead isolation kits for CD4 are always positive selection. But if you looked at the protocols for CD4 T cells, it's two steps. The first is negative selection for CD3+ cells and then a positive selection step for CD4.
You can't just use ONLY the CD4 selection beads, because this will also pick up DCs.
As for why most people perfer using the beads. It's less expensive (comparably), it's a lot faster, it's easier (you don't need to set gates), and you don't need a FACS sort.
Magnetic bead separation enrich your cell of interest by selecting for CD4 cells in this case. Therefore you can save time and increase purity of sorted cells by sorting something like a million T cells at 1000 cells per second with less than 50% of them going to waste instead of ten million cells at over 2000 per second and 80% going to waste because they are not even T cells.
Slower sorting speed increase purity and yield so you get more cells from each subset.
You can definitely do negative selection for T cells or CD4 Tcells. That way all other cells will be bound to beads and only the cell of interest will elute and untouched for your stimulation then analyse.
Positive selection uses antibodies for your cells of interest which could activate your cells in some matter making it a perturbation that confounds your question. Same applies to facs where antibodies is applied. In PBMC, such binding may activate your cells while sorting them. In enriched cells you are less likely to encounter this problem as less time is taken and it is ready for RNA analysis rather than having to stimulate them again after FACS.
Hi Rajeev.
In my opinion the better way is to isolate CD4 T cells with a negative selection kit.
I used to use the RosetteSep kit from Stemcell (https://www.stemcell.com/products/brands/rosettesep.html). You can isolate CD4 T cells directly from blood, following with a Ficoll procedure. You can find protocols and explanationson Stemcell website.
Good luck.
Hi Rajeev,
I would go for MACS, since you need to culture your CD4+ T cells; firstly because you will get more cells, and that is important since you will need to culture them in vitro and it is better to work with large numbers of cells in this case. As well, because it is better to start from naïve CD45RA+ CD4+ T cells, i.e, from cells not committed to a particular pathway of differentiation; and thirdly, unless your FACS purification is performed inside a biosafety cabinet (in order to prevent sample contamination), its better to do MACS to ensure that after purifications and cell culture you will not get bacterial cells instead of lymphocytes.
Good luck!
Francisco J Salgado
If you are interested in total CD4 T cells, I would suggest to use a CD4 T cell negative selection kit (e.g. Miltenyi or StemCell). If you are interested in untouched naive CD4 T cells you can first use a CD4 T cell negative selection kit and then purify naive cells by flow using CD45RO and CD57. The CD45RO/CD57 negative cells should be naive CD4 T cells. Of course you can add other activation markers, such as CD69 and HLA-DR, and chemokine receptors, such as CXCR3, CCR4 and CCR6, to be totally sure that you are selecting naive CD4 T cells and not EMRA CD4 T cells.
Hi
I suggest puré CD4 cell sorting, obviously you need to label with specific antobody. In comparison with MACS separation you will have better purity. If you go for MACS (positive ornegative selection) you shoul verify puré population by FACS.
bye
Do you want to differentiate the memory subsets (Th1, Th2, Th17) from naive CD4+ T cells in vitro or isolate them ex vivo? For the latter I would recommend FACS sorting after CD4+ MACS enrich utilizing naive/memory markers as well as Chemokine receptors as described by F. Sallusto et al. Article Heterogeneity of Human CD4(+) T Cells Against Microbes
Thanks everyone for your replies, as I am doing ex-vivo differentiation of the subsets, I have decided to use Dynabeads from thermofisher for the enrichment(I am aslo sampling RosetteSep from Stemcell Tech) for the enrichment of CD4 T cells.
David Bending Shen-An Hwang Vincent Oei Aurelie Schneider Francisco J Salgado Raffaello Cimbro Lucie Loyal
Why not Miltenyi? Rajeev Vikram
I know both Miltenyi and Dynabeads well. M has best yield, D is cheap. If you have the relevant magnet, then just stick to it then...
- Badges
- Science method