Journal: Bio-protocol

Article Title: Nuclei Isolation Methods on Frozen Clotted Blood Samples

doi: 10.21769/BioProtoc.5573

Figure Lengend Snippet: (A) Gating strategy for sorting singlets of B and T cells. (B) Sorted counts. Leukocytes were isolated from previously frozen blood clots and dissociated by following section A of this protocol. Cells were stained with an antibody panel targeting CD3, CD19, CD11b, and CD11c. Viability marker was not used due to interest in nuclei from subpopulations of non-viable leukocytes. After doublet exclusion and gating on height and area of cells, T-cell subsets were identified as CD3+ populations. B-cell subsets were identified as CD19+ populations. CD11b and CD11c were used to exclude monocytes. Single-stain controls were used for compensation. During this sort, we successfully sorted 57,894 T cells and 24,648 B cells. The full report is available in Supplementary material (Report S1).

Article Snippet: Flow cytometry antibodies (only applicable if flow sorting) a. FITC CD3 antibody (Miltenyi Biotec, catalog number: 130-113-700) b. PE CD19 antibody (Miltenyi Biotec, catalog number: 130-114-172) c. VioBlue CD11B (Miltenyi Biotec, catalog number: 130-110-616) d. APC CD11C (Miltenyi Biotec, catalog number: 130-114-110) e. Other antibodies (optional): Add other suitable antibodies to tailor the cell population of interest 3.

Techniques: Isolation, Staining, Marker