Figure 1.
Figure 1. Localization of proB and preB cells in relationship to IL-7– and CXCL12-producing cells. (A) Gating strategy for proB and preB cells. Left, BM cells gated on live CD19+ cells. Right, cKit expression distinguishes proB from preB cells. (B) Left, RAG2:GFP fusion protein expression in proB (red) and preB (blue) cells isolated from RAG2:GFP mice. Filled histograms show background fluorescence in wild-type preB cells. RAG2:GFP expression in proB (middle panel) and preB (right panel) in S phase (BrdU+) and in non–S phase (BrdU–). (C) Longitudinal 30-µm-thick femur section from lethally irradiated Il7GFP/+ Cxcl12dsRed/+ mouse reconstituted with RAG2:GFP BM cells stained to detect GFP (green), CD19 (cyan), and nuclei (DAPI, not shown). Cxcl12-dsRed fluorescent protein (red) was directly visualized. Data were pooled from two independent experiments (n = 2). (D) Quantification of proB (RAG2:GFPHi) and preB (RAG2:GFPLo) cells in contact with IL-7– and CXCL12-producing cells (see Materials and methods for details). Bars indicate average; circles indicate individual mice. (E) Quantification of colocalized RAG2:GFPHi (proB) and RAG2:GFPLo (preB) in contact with individual IL-7+ CXCL12+ cells (bottom right). Example of a RAG2:GFPHi (arrowheads) and a RAG2:GFPLo (arrows) cell in contact with the same IL-7+ cell. The same datasets were analyzed as in D. Error bars indicate SD. (F) Longitudinal 30-µm-thick section of a femur from a lethally irradiated Il7GFP/+ Cxcl12dsRed/+ mouse reconstituted with Rag1−/− (top panels) or Rag1−/− IghB1-8/+ (bottom panels) BM cells, stained to detect GFP (green) and CD19 (cyan). Cxcl12-dsRed was directly visualized (red). An entire femur/mouse was acquired; data were pooled from three independent experiments (n = 3). (G) Frequency of proB (Rag1−/−) and preB (Rag1−/− IghB1-8/+) cells in contact with IL-7+ CXCL12+ cells. Bars indicate average; circles depict individual mice. *, P < 0.05 by unpaired Student’s t test. Scale bars, 50 µm. Data in all panels are representative of two to five independent experiments.

Localization of proB and preB cells in relationship to IL-7– and CXCL12-producing cells. (A) Gating strategy for proB and preB cells. Left, BM cells gated on live CD19+ cells. Right, cKit expression distinguishes proB from preB cells. (B) Left, RAG2:GFP fusion protein expression in proB (red) and preB (blue) cells isolated from RAG2:GFP mice. Filled histograms show background fluorescence in wild-type preB cells. RAG2:GFP expression in proB (middle panel) and preB (right panel) in S phase (BrdU+) and in non–S phase (BrdU–). (C) Longitudinal 30-µm-thick femur section from lethally irradiated Il7GFP/+ Cxcl12dsRed/+ mouse reconstituted with RAG2:GFP BM cells stained to detect GFP (green), CD19 (cyan), and nuclei (DAPI, not shown). Cxcl12-dsRed fluorescent protein (red) was directly visualized. Data were pooled from two independent experiments (n = 2). (D) Quantification of proB (RAG2:GFPHi) and preB (RAG2:GFPLo) cells in contact with IL-7– and CXCL12-producing cells (see Materials and methods for details). Bars indicate average; circles indicate individual mice. (E) Quantification of colocalized RAG2:GFPHi (proB) and RAG2:GFPLo (preB) in contact with individual IL-7+ CXCL12+ cells (bottom right). Example of a RAG2:GFPHi (arrowheads) and a RAG2:GFPLo (arrows) cell in contact with the same IL-7+ cell. The same datasets were analyzed as in D. Error bars indicate SD. (F) Longitudinal 30-µm-thick section of a femur from a lethally irradiated Il7GFP/+ Cxcl12dsRed/+ mouse reconstituted with Rag1−/− (top panels) or Rag1−/− IghB1-8/+ (bottom panels) BM cells, stained to detect GFP (green) and CD19 (cyan). Cxcl12-dsRed was directly visualized (red). An entire femur/mouse was acquired; data were pooled from three independent experiments (n = 3). (G) Frequency of proB (Rag1−/−) and preB (Rag1−/− IghB1-8/+) cells in contact with IL-7+ CXCL12+ cells. Bars indicate average; circles depict individual mice. *, P < 0.05 by unpaired Student’s t test. Scale bars, 50 µm. Data in all panels are representative of two to five independent experiments.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal