linearflow™ green flow cytometry intensity calibration kit  (Thermo Fisher)

Journal: The Journal of Clinical Investigation

Article Title: Lineage-affiliated transcription factors bind the Gata3 Tce1 enhancer to mediate lineage-specific programs

doi: 10.1172/JCI83894

Figure Lengend Snippet: (A) Two CRISPR/Cas9 plasmids expressing gRNAs that correspond to sequences surrounding the 7.1 kbp that define the boundaries of Tce1 were coinjected into mouse fertilized oocytes. Tce1-deleted mutant allele founder (F0) mice were intercrossed to obtain homozygous deletion mutant mice. (B) Thymocytes, (C) peripheral blood cells, and (D) splenocytes isolated from F2 animals at 5 to 6 weeks of age bearing homozygous (white circle) or heterozygous (black circle) deletions of Tce1 were analyzed for cell surface expression of T cell stage–specific markers. The absolute numbers of Lin–cKithiCD25– (ETP), Lin–cKithiCD25+ (DN2), Lin–cKitlo/–CD25+ (DN3), Lin–cKitlo/–CD25– (DN4), CD4+CD8+ (DP), CD4+CD8–CD3+TCRβ+ (CD4 SP), and CD4–CD8+CD3+TCRβ+ (CD8 SP) thymocytes are shown at the top. Each stage of T cells was isolated by flow cytometry and analyzed for the expression of Gata3 mRNA, as normalized to Hprt by qRT-PCR. Each circle represents an individual mouse, and black bars represent the average for each genotype. Data are representative of the summary of 2 independent experiments. *P < 0.05 by Student’s t test.

Article Snippet: For every experiment in which EGFP expression was analyzed, a LinearFlow Green Flow Cytometry Intensity Calibration Kit (Molecular Probes) was used.

Techniques: CRISPR, Expressing, Mutagenesis, Isolation, Flow Cytometry, Quantitative RT-PCR