Laser-Based Microdissection of Single Cells from Tissue Sections and PCR Analysis of Rearranged Immunoglobulin Genes from Isolated Normal and Malignant Human B Cells

Recent advances have enabled mapping of histone modifications in single cells, but current methods are constrained to profile only one histone modification per cell. Here we present an integrated experimental and computational framework, scChIX (single-cell chromatin immunocleavage and unmixing), to map multiple histone modifications in single cells. We first validate this method using purified blood cells and show that although the two repressive marks, H3K27me3 and H3K9me3, are generally mutually exclusive, the transitions between the two regions can vary between cell types. Next we apply scChIX to a heterogenous cell population from mouse bone marrow to generate linked maps of active (H3K4me1) and repressive (H3K27me3) chromatin landscapes in single cells, where coordinates in the active modification map correspond to coordinates in the repressive map. Linked analysis reveals that immunoglobulin genes in the region are in a repressed chromatin state in pro-B cells, but become activated in B cells. Overall, scChIX unlocks systematic interrogation of the interplay between histone modifications in single cells.

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Characterization of Ryanodine Receptor–mediated Calcium Release in Human B Cells

Anesthesiology ◽

10.1097/00000542-200606000-00014 ◽

2006 ◽

Vol 104 (6) ◽

pp. 1191-1201 ◽

Cited By ~ 17

Author(s):

Leslie C. McKinney ◽

Thomas Butler ◽

Shawn P. Mullen ◽

Michael G. Klein

Keyword(s):

B Cells ◽

Malignant Hyperthermia ◽

B Cell ◽

Cell Lines ◽

Single Cells ◽

Human B Cells ◽

Malignant Hyperthermia Susceptible ◽

Increased Sensitivity ◽

Normal Human ◽

Human B Cell

Background Mutations in the ryanodine type 1 receptor (RyR1) are causative for malignant hyperthermia. Studies in human B lymphocytes have shown that measurement of RyR1-mediated intracellular Ca(2+) (Ca(2+)(i)) release can differentiate between normal and malignant hyperthermia-susceptible individuals. The authors have further developed the B-cell assay by pharmacologically characterizing RyR1-mediated Ca release in two normal human B-cell lines and demonstrating increased sensitivity of lymphocytes to the RyR1 agonist 4-chloro-m-cresol (4-CmC) in the porcine model of MH. Methods Ca(2+)(i) was measured fluorometrically using fura-2 in populations of cells in suspension or with fluo-4 in single cells using confocal microscopy. The Dakiki and PP normal human B cell lines were used, as well as lymphocytes obtained from normal and malignant hyperthermia-susceptible pigs. 4-CmC was used to elicit RyR1-mediated Ca release; all experiments were performed in the absence of external Ca(2+). Results EC(50) values for 4-CmC were 0.98 and 1.04 mm for Dakiki and PP cells, respectively, demonstrating reproducibility. The 4-CmC-induced increase in Ca(2+)(i) was eliminated by thapsigargin and was unaffected by xestospongin C. The Ca(2+)(i) increase was separable from mitochondrial stores and was inhibited by azumolene. Caffeine did not induce Ca(2+)(i) release, but ryanodine depleted intracellular stores by 50%. Lymphocytes from pigs carrying the Arg614Cys mutation in RyR1 showed increased sensitivity to 4-CmC (EC(50) = 0.47 vs. 0.81 mm for cells derived from normal animals). Conclusions RyR1-mediated Ca(2+) signals can be pharmacologically distinguished from other intracellular sources in human B cells, and alterations of RyR1 function can be successfully detected using Ca(2+) release from intracellular stores as an end point.

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