Impaired IGH locus compaction in cultured CD19⁺CD127⁺ and CD19⁺CD127⁻ cells. (A) Schematic representation of the human IGH locus and the combinations of bacterial artificial chromosome clones used as 3D FISH probes are shown. The distance separating each of the 3 probes and their positions within the IGH locus were determined from the IMGT database³⁵  Distal V_H probes were labeled with Alexa Fluor 568 (red), proximal V_H probes with Cy5 (blue), and C_H probes with Alexa Fluor 488 (green). Numbers in the rectangles represent the size of the regions recognized by the probes and the numbers below the arrows represent genomic distances between probe sets. (B-D) Scatter plots showing the distances in micrometers (y-axis) separating distal V_H, proximal V_H, and C_H regions in FACS-purified pediatric BM pro-B and small pre-B cells, CD8⁺ thymocytes, and FACS-purified CD19⁺CD127⁺ and CD19⁺CD127⁻ populations from xenogeneic cultures. Except for pro-B cells (1 donor) at least 2 different donors and at least 100 alleles were analyzed per population. Red horizontal lines represent median distances between indicated probes in each population. The nonparametric Mann-Whitney test was used to calculate significance levels between paired populations (horizontal bars). *P < .05; **P < .01; ***P < .001. (E) Representative images of IGH loci in the primary and cultured cells populations captured with an SP5 confocal microscope (Leica Microsystems). Using a 63×/1.4 NA lens, images of ∼ 70 serial optical sections spaced by 0.148 μm were acquired at room temperature using LAS AF 1.8.2 software (Leica Microsystems). The datasets were deconvolved and analyzed with Huygens Professional software (Scientific Volume Imaging).