Figure 6.
Bone marrow progenitor profile of SLFN14K208N/+mice. (Ai) Representative images of H&E-stained femur sections from SLFN14K208N/+ and SLFN14+/+ mice. Femurs were fixed in 4% formaldehyde and decalcified before sectioning, staining and quantification of MK number per field of view. MKs are indicated by arrowheads. Scale bars, 50 µm. (Aii) Quantification of MK number per field of view from 3 mice per genotype. Two femurs per mouse were sectioned, and 10 to 13 fields of view per section were quantified blind. Students t test was used to assess significance. (B) Quantification of MKs in bone marrow. (Bi) MK staining: MKs were identified by CD41 (αIIb) allophycocyanin (APC) and CD42 (GPIb) fluorescein isothiocyanate (FITC) double staining. (Bii) Quantification of MKs in whole bone marrow by flow cytometry. (C) Quantification of erythroid progenitors in bone marrow. (Ci) ProE staining: ProEs were identified as double-positive CD71 (transferrin receptor 1) phycoerythrin (PE) and Ter119 FITC cells (ProE gate). Maturation of ProEs to mature erythrocytes can be monitored by Ter119hi expression and loss of CD71 expression (EryB and EryC gates). Note the spread of intermediate cells in EryBs, supporting evidence for an altered EryB fate in heterozygotes. (Cii) Quantification of erythroid progenitors in the bone marrow. (Ciii) Confocal images of flow cytometry samples show a double-positive ProE population. Ter119 Alexa Fluor 488 and CD71 Alexa Fluor 647 and DAPI counterstain. Scale bars, 50 µm. (D) Quantification of MEPs in bone marrow. (Di) MK-EB–primed MEPs: MEPs were identified as a small population positive for CD71 (transferrin receptor 1) PE and CD41 (αIIb) APC (MEP). Flow cytometry plots show a slight, but insignificant, increase in MEP cell numbers in SLFN14 K208N mice compared with wild-types. (Dii) MEP quantification in the bone marrow. Staining for these markers highlights MEP cells preferential to the MK or EB lineage. (Diii) Representative images of CD41 Alexa Fluor 488 and CD71 Alexa Fluor 647 stained bone marrow cells imaged by confocal microscopy and using DAPI counterstain. Scale bars, 50 µm. All bone marrow flow cytometry data and quantifications are representative of 4 to 6 mice per genotype/staining condition.

Bone marrow progenitor profile of SLFN14K208N/+mice. (Ai) Representative images of H&E-stained femur sections from SLFN14K208N/+ and SLFN14+/+ mice. Femurs were fixed in 4% formaldehyde and decalcified before sectioning, staining and quantification of MK number per field of view. MKs are indicated by arrowheads. Scale bars, 50 µm. (Aii) Quantification of MK number per field of view from 3 mice per genotype. Two femurs per mouse were sectioned, and 10 to 13 fields of view per section were quantified blind. Students t test was used to assess significance. (B) Quantification of MKs in bone marrow. (Bi) MK staining: MKs were identified by CD41 (αIIb) allophycocyanin (APC) and CD42 (GPIb) fluorescein isothiocyanate (FITC) double staining. (Bii) Quantification of MKs in whole bone marrow by flow cytometry. (C) Quantification of erythroid progenitors in bone marrow. (Ci) ProE staining: ProEs were identified as double-positive CD71 (transferrin receptor 1) phycoerythrin (PE) and Ter119 FITC cells (ProE gate). Maturation of ProEs to mature erythrocytes can be monitored by Ter119hi expression and loss of CD71 expression (EryB and EryC gates). Note the spread of intermediate cells in EryBs, supporting evidence for an altered EryB fate in heterozygotes. (Cii) Quantification of erythroid progenitors in the bone marrow. (Ciii) Confocal images of flow cytometry samples show a double-positive ProE population. Ter119 Alexa Fluor 488 and CD71 Alexa Fluor 647 and DAPI counterstain. Scale bars, 50 µm. (D) Quantification of MEPs in bone marrow. (Di) MK-EB–primed MEPs: MEPs were identified as a small population positive for CD71 (transferrin receptor 1) PE and CD41 (αIIb) APC (MEP). Flow cytometry plots show a slight, but insignificant, increase in MEP cell numbers in SLFN14 K208N mice compared with wild-types. (Dii) MEP quantification in the bone marrow. Staining for these markers highlights MEP cells preferential to the MK or EB lineage. (Diii) Representative images of CD41 Alexa Fluor 488 and CD71 Alexa Fluor 647 stained bone marrow cells imaged by confocal microscopy and using DAPI counterstain. Scale bars, 50 µm. All bone marrow flow cytometry data and quantifications are representative of 4 to 6 mice per genotype/staining condition.

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