Hi everyone!
I have trouble isolating frozen PBMCs with CD25+CD49d– Regulatory T Cell Isolation Kit, human (Miltenyi, # 130-094-551). It is recommended to work with fresh cells, but I have a limited number of cryos of the rare patient group and I have to start isolation with 20-30 million cells in total.
I follow the manufacturer's protocol. However, in order to increase the purity, I extend the incubation times by 10 minutes since I have done it in an ice-filled box at +4C (I do this application because I've seen that it increases the purity in the isolations previously made with CD19 microbeads and I achieved 90-95% purity ).
In my last experiment, I started with 22 million cells, after cells passed through LD column, there were 750,000 cells left (according to the immunophenotyping result of the healthy donor, there should be approximately 2.5 million CD25+ T cells). When I selected the cells I marked with the CD25 microbead in step 2 with the MS column, I had 30,000 cells left. Some even less than that.
In addition, when viewed by flow cytometry and microscopy, cells passing through the LD column are very viable, while only a few (1-5%) of them are alive after isolation with the MS column. I tried with the column with a different lot number in case the problem was caused by the column, but the result did not change.
I will do RNA isolation and qPCR from the isolated cells, but the RNA isolation I made with the amount I obtained did not work well.
Do you have any suggestions? Many thanks in advance.
Dear Elif Sanli ,
From the above text, I do not know whether you have tested the kit with fresh PBMC. My recommendation is that you first test the whole procedure with fresh PBMC samples of healthy donors.
When you have confirmed that everything went well with fresh PBMC and the kit, then you may begin working with the patient samples to compare the results with those of fresh PBMC.
By doing so, you can distinguish issues associated with the kit and issues associated with the frozen patients sample. This would disambiguate your problems and ease your troubleshooting accordingly.
Dear Elif Sanli
Please see the attached publication for a detailed thawing, dead cell removal and staining procedure protocol for lymphocyte enrichment via MACS from frozen PBMCs. As you're indicating you want to use your cells for (bulk) qPCR their suggestions for an optimized protocol for single cell sequencing should help.
Article An optimized workflow for single-cell transcriptomics and re...
All the best & good luck with your experiment,
Michael
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