Hi,
I am optimizing a flow protocol for LS-180 cell line, which can not be trypsinized. First experiment wend suboptimal due to detritus and doublets Any trips or tricks for making this cell line flowing?
Guillem Argilés I'm not an expert on this cell line but my own cell lines cannot be trypsinized either. Instead, I use TrypLE - this is a gentle cell dissociation reagent that replaces trypsin. Use in exactly the same way as trypsin.
https://www.thermofisher.com/uk/en/home/life-science/cell-culture/mammalian-cell-culture/reagents/trypsin/tryple-express.html?icid=cvc-dissociation-c2t2
Dear Guillem!
Please You also look at this article:
Article Mesenchymal stromal cells induce epithelial-to-mesenchymal t...
Tumor cell lines
Established human CRC cell lines (HCT116, LS180, COLO205, HT29 and SW480) were purchased from European Collection of Cell Cultures (ECACC, Salisbury, UK).
Flow cytometric analysis and cell sorting
Phenotypes of expanded MSC were analyzed upon staining with the following antibodies: allophycocyanin (APC)-labeled anti-CD34 (clone 581), anti-CD90 (clone 5E10), phycoerythrin (PE)-labeled anti-CD31 (clone WM59), anti-CD73 (clone AD2), anti-CD44 (clone G44-26), anti-CD29 (clone MAR4), fluorescein-isothiocyanate (FITC)-labeled anti-CD45 (clone 2D1) (all from BD Biosciences, San Jose, CA) and anti-CD105 (clone SN6, AbDSerotec, Raleigh, NC). For the analysis of CRC cells in coculture with stromal cells, the following antibodies were used: APC-labeled anti-EpCAM (clone EBA-1), FITC-labeled anti-CD90 and PE-labeled anti-CD44, anti-CD166 (all from BD Biosciences) or anti-CD133 (Miltenyi Biotec, Auburn, CA) or PE-labeled anti-TGF-β (clone 9016, R&D Systems). Propidium iodide (PI, 0.5 µg/mL) was added to all samples prior to analysis. Samples were analyzed by a dual laser BD FACS Calibur flow cytometer (BD Biosciences), following exclusion of dead cells based on PI incorporation. Analysis was performed using FlowJo software (Tree Star, Ashland, OR). For sorting of tumor cells and MSC from cocultures, cells were stained with APC-labeled anti-EpCAM and FITC-labeled anti-CD90. Dead cells were excluded based on DAPI incorporation. Cell sorting was performed using a BD Influx cell sorter (BD Biosciences). Purity of sorted cells was ≥98%.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line