We have a done a comparison between mechanical and enzymatic dissociation and have noticed better viability with enzymatic dissociation. Dead cell removal kits may work but only to an extent. I wouldn't use column based separation methods as dead cells are sticky and would stick to your column and take live cells along with them. Maybe switch to a bead based separation system (Stem Cell Technologies has one). You need to have a good proportion of viable cells in the initial cell suspension for you to achieve the level of dead cell removal you are aiming for. Maybe a two step protocol might work better.

1. Do a Ficoll separation to remove dead cells and improve % viability

http://www2.egr.uh.edu/~nvaradar/private/Prots/Fico_LD.pdf

2. Then use a dead cell removal kit (bead based) to further improve % viability

an old protocol is: pellet your cells, add 2ml trypsin (same you use for detach cells, you can increase if huge pellet)+DNASI 2000U 2min rt pipetting up and down. then wash with your wash buffer. Dead cells will go away, but it is not useful for debris...good luck!

Hi,

You may try to FACS-sort your cells with addition of DAPI. The dead cells will be DAPI-positive, so you can gate them out. You may also try to stain them with PI, if you have DAPI channel occupied with Hoescht (for example).

We always use FACS sorted cells for scRNA-Seq approaches, because the method is quite expensive and it is better to have the best cells as the input.

Best,

Michail

Hi Mayra,

If you have a lot of debris and dead cells after dissociation, dead cell removal reagents probably are not sufficient to get rid of them. I would suggest to use the Debris Removal Solution fom Miltenyi. This is very efficient to remove all the debris generated after dissociation of adult mouse brain or mouse heart.

https://www.miltenyibiotec.com/DE-en/products/macs-sample-preparation/removal-reagents/debris-removal-solution.html#130-109-398

But perhaps the dissociation step here needs further optimization so that you don´t get so many debris and dead cells. With which kind of tumor samples are you working with? The toughness of the tumor and presence of fibrosis or necrotic parts can be challenging for the dissociation and lead to low yields and cell viability. I have some experience regarding tumor dissociation, in case you have further questions.

Best,

Maria

Hi, 9 months passed since you have asked the question. So how is it going with you single cells sequencing? Did you succeed in preparing high quality single cell suspension? I have trouble in the sample preparation too. Would you mind sharing your successful solution? Truly apprecitate that.

My experience with brain and liver-infiltrating Lymphocytes and now in melanoma-infiltrating lymphocytes has taught me to do gently enzymatic dissociation followed by removal of dead cells with 30% percoll cushions. That works for me for downstream observations like RNA-seq and ex vivo stimulation. Good luck

Hi Mayra,

I wanted to ask whether you finally succeeded in enrichment of viable cells.

Which protocol would you recommend to use, if you work with primary tumors?

Best; N

I guess, since it is for single cell sequencing, quantity is not an issue but quality is? One way to distinguish really healthy cells from damaged but intact ones plus separation from debris is to let cells briefly - 1-3 hours should be sufficient - attach to a conventional or coated culture dish, wash carefully and then re-trypsinize. Of course, this last step will to some extent compromise viability but the cell batch will have originated from the healthiest portion of the total sample. We used this procedure for related purposes.

Hi everyone,

Sorry, I haven't updated the post. Many thanks for all suggestions!

I have tested some of these protocols and the best was to follow MACS Miltenyi recommendation.

I have successfully performed the scRNA-seq! I dissociate human tumors using the GentleMACS system: the instrument gentleMACS™ Dissociator (the one without heating - code 130-093-235) and enzymes from the tumor dissociation kit (code 130-095-929). I contacted the Miltenyi support team and they suggested an additional treatment with nuclease after obtaining the single-cell suspension. I noticed that this step is now described in the datasheet of the kit (step 15 in the protocol). After dissociation, I removed the dead cells using the dead cell removal kit from MACS Miltenyi as well (code 130-090-101). I have tested the dead cell removal from StemCell but it is not efficient as Miltenyi. I think a critical point is this nuclease treatment as well as the filtering step prior to applying cell suspension onto the collum (Pre-Separation Filters (70 µm) - MACS Miltenyi). Just to make it more clear, I summarize here the steps: - tissue dissociation (gentleMACS dissociator and tumor dissociation kit) - Filter single-cell suspension: MACS SmartStrainers (70 µm) - Nuclase treatment - Dead cell removal (MACS Miltenyi kit): remember to pass the cell suspension through a pre-filter (code 130-095-823) prior to applying onto the column.

To finish, it looks like I work for MACS Miltenyi but I don't! I compared their products with selection kits from STEMCELL and this last company really didn't work on my tissue. Please, let me know if you need other help.

I hope it will work for you as well, Norbert Schweifer

Hi Mayra, have you tried an FCS gradient centrifugation? In cell culture you remove sometimes debris and dead cells by do so.

In a 15 mL tube just layer underneath your cell suspension approx. 5 mL FCS. Spin 10 min 1200 rpm w/o brake. Debis and dead cells are on top of the interphase. Carefully remove the supernatant by aspiration, then resuspend remaining cells in medium. (it analogous to blood separation by ficol)

Hi Mayra Carneiro !

I am trying to magnetically sort tumor associated endothelial cells for ATAC seq. I need at least 50,000 cells at atleast 90% viability. Can you tell me what percentage viability and number of cells after the protocol you have mentioned?

Thank you!

Hello Ingo Fricke, Thank you for sharing. I have a similar problem as the original post where I have a lot of cellular debri with my dissociated tumor cells. I want to remove the cellular debri without the use of kits. I googled FCS gradient centrifugation and got a lot of confusing posts. Specifically I want to know do you mean Fetal Calf Serum (FCS) or is there a reagent called FCS maybe Ficoll based? Can you share a detailed protocol?

I heard the other way around but I am not sure 100%. Centrifuge at 800-1000xg for 3-5 minutes, so that nearly almost dead cells will burst and will be in the supernatant. might not be logical but if anyone can explain better, I would be appreciated. I always tried to use 300-400xg to remove dead cells before.