Figure 4.
Figure 4. Mepacrine labels acidic endolysosomes in MKs. Murine fetal liver-derived MKs (FL-MKs) (A,D-E), G1ME2 cells differentiated for 3-4 days to MKs (G1ME2-MK) (B,D-E), or human CD34-positive HPC-derived MKs (CD34+ MK) (C-E) were incubated with 50 μM mepacrine (green) and 200 nM LysoTracker Red DND-99 (red) for 30 min; cells in panel B were also incubated with Alexa Fluor-647-conjugated anti-CD41 antibody. Images are of a single plane; panel C is deconvolved. In panels A and C, a corresponding DIC image is shown, and the cell outline is indicated by the dotted line (A-C). In panel B, CD41 labeling is also shown; ×2 magnification of boxed regions (insets); examples of overlap between mepacrine and LysoTracker (arrowheads). Scale bars, 5 μm. (D) The number (mean ± SD) of puncta per platelet labeled by mepacrine alone (#mep-only), LysoTracker Red alone (#LyTr-only), or both (#coloc.) in G1ME2-MK (n = 33), FL-MK (n = 30), and CD34+ MK (n = 43). (E) Quantification of the percentage (mean ± SD) of mepacrine labeling that colocalized with LysoTracker Red labeling in each cell type (n = 33 G1ME2-MK; n = 30 FL-MK; n = 43 CD34+ MK). Values are normalized to 100% for overlap of LysoTracker Red with LysoTracker Green DND-26 in G1ME2-MK (n = 79). Fetal-liver MKs were imaged by spinning-disk confocal microscopy at room temperature with an Olympus IX71 inverted microscope equipped with a Hamamatsu ImagEM EM-CCD camera, a 60× Plan Apo water immersion objective (1.2 NA), and MetaMorph software. G1ME2-MKs were imaged by spinning-disk confocal microscopy at room temperature with an Ultraview inverted microscope equipped with a 63× Plan Apochromat lens, a Hamamatsu Orca-ER CCD camera, and Volocity software. CD34+ MKs were imaged by wide-field microscopy at room temperature with a Leica DM IRBE equipped with a 100× Plan Apochromat objective lens (1.4 NA), a Hamamatsu Orca Flash 4 CMOS camera, and Leica Application Suite software. Imaging medium was IMDM. Mepacrine has an excitation/emission of 436/525 nm; LysoTracker Red excitation/emission: 577/590 nm. LyTrG, LysoTracker Green; LyTrR, LysoTracker Red.

Mepacrine labels acidic endolysosomes in MKs. Murine fetal liver-derived MKs (FL-MKs) (A,D-E), G1ME2 cells differentiated for 3-4 days to MKs (G1ME2-MK) (B,D-E), or human CD34-positive HPC-derived MKs (CD34+ MK) (C-E) were incubated with 50 μM mepacrine (green) and 200 nM LysoTracker Red DND-99 (red) for 30 min; cells in panel B were also incubated with Alexa Fluor-647-conjugated anti-CD41 antibody. Images are of a single plane; panel C is deconvolved. In panels A and C, a corresponding DIC image is shown, and the cell outline is indicated by the dotted line (A-C). In panel B, CD41 labeling is also shown; ×2 magnification of boxed regions (insets); examples of overlap between mepacrine and LysoTracker (arrowheads). Scale bars, 5 μm. (D) The number (mean ± SD) of puncta per platelet labeled by mepacrine alone (#mep-only), LysoTracker Red alone (#LyTr-only), or both (#coloc.) in G1ME2-MK (n = 33), FL-MK (n = 30), and CD34+ MK (n = 43). (E) Quantification of the percentage (mean ± SD) of mepacrine labeling that colocalized with LysoTracker Red labeling in each cell type (n = 33 G1ME2-MK; n = 30 FL-MK; n = 43 CD34+ MK). Values are normalized to 100% for overlap of LysoTracker Red with LysoTracker Green DND-26 in G1ME2-MK (n = 79). Fetal-liver MKs were imaged by spinning-disk confocal microscopy at room temperature with an Olympus IX71 inverted microscope equipped with a Hamamatsu ImagEM EM-CCD camera, a 60× Plan Apo water immersion objective (1.2 NA), and MetaMorph software. G1ME2-MKs were imaged by spinning-disk confocal microscopy at room temperature with an Ultraview inverted microscope equipped with a 63× Plan Apochromat lens, a Hamamatsu Orca-ER CCD camera, and Volocity software. CD34+ MKs were imaged by wide-field microscopy at room temperature with a Leica DM IRBE equipped with a 100× Plan Apochromat objective lens (1.4 NA), a Hamamatsu Orca Flash 4 CMOS camera, and Leica Application Suite software. Imaging medium was IMDM. Mepacrine has an excitation/emission of 436/525 nm; LysoTracker Red excitation/emission: 577/590 nm. LyTrG, LysoTracker Green; LyTrR, LysoTracker Red.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal