![]() ![]() A FACS sort was performed based on CD34 and CD25 expression resulting in two subclones: subclone 1 CD34 +CD25 + and subclone 2 CD34 +CD25. Plasma membrane protein marker CD25 was identified as most distinguishing marker. (C) A Principal Component analysis was performed using aberrantly expressed plasma membrane protein markers, showing the most distinguishing markers for Principal Component 1 (PC1) and Principal Component 2 (PC2). (A and B) (A) Gating strategy for the sorting of subpopulations (B) Expression of plasma membrane protein markers of an AML sample plotted together with the expression of these markers in a healthy control in order to identify which plasma membrane protein markers are aberrantly expressed. Outcome of subclone identification by Principal Component analysis ![]() Data analysis and the thawing of a second AML sample for sorting can be done on a different day. Other flow cytometers may also be used if the lasers are sufficient for the used fluorophores. Note: We acquire flow data at the MacsQuant Analyzer 10. The ranking of these markers was based on the number of times it was positive in a cohort of 87 AML samples and on literature describing the use of specific markers in AML. In case of limited patient material, for instance at follow-up time points, a prioritized list consisting of 15 markers can be used, indicated by an asterisk behind the antibody in the key resources table. If desired, PI (at 1 μg/mL) can be added to the backbone panel in order to discriminate viable cells. The largest cell loss during the thawing procedure is cleaned by the DNase in the NCS mix and does not result in the accidental staining of large number of dead cells. Note: The percentage of viable, thawed cells when measuring the marker panel usually lies between 70-90%. Note: While we have used a backbone panel with antibodies against CD34, CD38, CD45 and CD45RA, other markers, or additional markers such as CD64 or CD117, can be included as backbone markers as well. For staining of cells in a 96-well plate some adjustments may be needed in the protocol. Note: The steps described above have been optimized for staining of cells in FACS tubes. It can serve as back-up if a pipetting mistake is made with the marker panel. Keep this at 4☌ until all PE-labeled marker panel antibodies have been added. Note: The extra volume of backbone-stained cells will be approximately 300 μl. National Center for Biotechnology InformationĪcquire expression data using a flow cytometer Chemicals, peptides, and recombinant proteins ![]()
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