Dear Hamid Reza; What if it reacts with several cells, two or more?

You could approach the question by a single cell sorting technique:

You label your lung digest (single cell suspension, filter on a 40 um cell strainer) with the markers of interest food your fibroblasts. You also add markers for the cells that you want to exclude: Ly6C for your monocytes for example. Then, you make your gating strategy as follow: Non-Debris cells, Singlets (FSC-W SSC-W or else), Alive (DAPI neg or other live/dead). From there, you select cells that are negative for the undesired cells: Ly6C-... Following that, you make sure that you are sorting the fibroblasts by positively gating them through their specific markers.

For the sort, use a single cell sorting in 96wp. This will give you your clonality.

I hope it helps you.

Dear Philippe;

How we can confirm that final isolated cells are purely fibroblasts. Then would you please explain a bit more about single cell sorting in 96wp?

Or if you have heard anything about other techniques for single cell separation, besides MACS and FACS. Also we have biopsies. I mean that very small pieces and actually very limited number of cells.

Dear Sadegh,

I'll answer in three parts:

1a. How to confirm that they are really fibroblasts? Well, this would be difficult if you do a single cell sorting as the cell is then directly deposited in a well. But upon expansion, it will be easy to analyze the daughter cells again by flow cytometry and confirm their lineage with your specific markers. But a priori, it should be fine, as you selected these cells based on their specific markers and the absence of markers of other cells. This implies sorting more than one plate, of course...

1b. Explanation on single cell sorting: Essentially, single cell sorting requires a FACS that can deposit the selected cell directly in the well of the 96wp. This is done by selecting your cells by a specific gating as I described previously. Once a cell is deposited in a well, the sorter will change well and deposit a fibroblast in it when one is encountered. The expected result is NOT one fibroblast per well, but actually less as some will die... This is why you should prepare many plates.

2. Other techniques. You will have to exclude other cells, regardless of the technique that you want to employ in order to generate clones (cultures issued from one cell). MACS, FACS... The more specific you are at the beginning, the better result you get at the end. Alternatively, you could decide to let your sample (digested to single cell, erythrocytes lysed...) adhere for 4h and waste the floating cells. The adherent cells will be enriched (not pure!!!) fibroblasts. Then you can do a limiting dilution

3. What is a limiting dilution? You count the cells that you get (MACS or if adhesion step, after trypsin retrieval). Then you resuspend them in a volume where you get 1 cell/200 ul and seed 200 ul/well of a 96wp. Of course, this is not perfectly precise, as sometimes, you will have 0 or 2 cells / well. Depending of what you really want (if clonality if of critical importance or not), you can also resuspend so that you have 0,5 cell/200 ul, so that you do not start it with 2 cell/well, but obviously, many wells will contain nothing. Then, you let them grow and confirm their fibroblast phenotype by FCM later.

I hope it helps!

Dear Philippe;

really clear answers. Thanks. There is a limitation that I don't want to expand cells. I want to isolate fibroblast directly from biopsies. Then lysing the collected cells for cDNA preparation and microarray analysis. Then I should be able to collect appropriate and pure collection of fibroblasts. Expansion will change the expression profiling of the cells. Microarray has single cell resolution but i cant trust. Because every fibroblast cell has a especial condition within the tissue. One may be in G1 and the other may be in G2. they are different. So I should use a collection of cells in order to minimize in one tissue differences.

I know it has been long.

Can you share your isolation protocol? Where you able to successfully isolate fibroblasts?