|
|
Blood, Vol. 114, Issue 2, 290-298, July 9, 2009
Altered cellular dynamics and endosteal location of aged early hematopoietic progenitor cells revealed by time-lapse intravital imaging in long bones
Blood Köhler et al.
114: 290
Supplemental materials for: Köhler et al
Files in this Data Supplement:
- Document 1. Mathematical analysis of cell surface area and cell volume based on image data: description of image analysis procedure (PDF, 75.1 KB)
- Figure S1. Demonstrating the process of image reconstruction for the measurement of distances (JPG, 419 KB)
-
We show this here for an example, where the bone structure is visualized by means of its autofluorescence. Imaging the bone surface by means of SHG simplifies the procedure, as there is hardly any overlap of the bone signal in the fluorescence channel. The values shown in brown/green depict the numbers that we adjusted in the relative image controls of Volocity to obtain the result shown in the figure. Of note: in step 5 only the black level of the bone channel is adjusted, while the fluorescence channel of the cell remains unchanged, because this immediately changes the size of the cell and would strongly influence the distance measurement as well as measurements on cell motility. The two large cells under 5 and 6 are magnifications of the area boxed in 5 to demonstrate the process of measurement as well as the presence (5, dashed line depicts bone median plane) or absence (6, white arrow) of the brown bone signal, if the bone channel is not adjusted properly. The median plane of the bone is taken as a reference for the distance measurement. The measured value for the shown cell is depicted. Abbreviations: B (brightness), BL (black level), BP (black point), D (density), G (gamma), WP (white point), autofl. (autofluorescence channel), CFSE fl. (fluorescence of the green CFSE channel).
- Figure S2. Validation of experimental methods (JPG, 140 KB)
-
(A) To demonstrate that measurements of the distance of L-S+K+ cells to the endosteum are not influenced by the CTO or CFSE staining protocols, we determined the individual distance values obtained for analogous preparations of HSC stained with either CFSE or CTO. The data demonstrate no significant difference in the two approaches, confirming that the staining with either CFSE or CTO has no significant (p= 0,4843) influence on the measured distance value. n=5, horizontal bars represent mean. (B) CAFC adhesion assays were performed to demonstrate validity. Cells were seeded on FBMD-1 stroma cells and the washed off cells (2, 3, and 4 hours adhesion) collected and seeded onto another layer of FBMD-1 cells. The frequency of CAFC day 7 cells was then determined and the frequency of the adhesive and the less-adhesive (non-adherent) cells added and compared to the CAFC frequency determined by a CAFC assay in the identical, non-treated BM sample (control). Adherent and non-adherent cells add up to close to 100% with respect to the untreated control (average value for 2, 3, and 4 hours equals 101% ± 6%). The CAFC adhesion assay is thus suitable to determine the adhesion abilities to stroma cells of functionally defined primitive hematopoietic progenitor cells. n=4 based on two biological repeats. Shown are mean values + 1 SEM.
- Figure S3. Osteoblasts (green) at or close to the endosteum (brown/red) can be visualized in animals bearing a YFP transgene under the control of the 2.3kb Col1a promoter (JPG, 65.7 KB)
-
- Video 1. Unfractionated bone marrow (BM) cells were labeled with either CFSE or CTO according to standard protocols and co-transplanted at 106 each into recipient animals (MPG, 10.4 MB)
-
Fluorescently labeled cells inside the bone cavity were analyzed by MP-IVM 16 hours post transplantation as described in Fig. 1 and Document 1.
- Video 2. Visualization of macrophages and dendritic cells residing in bone marrow (MPG, 5.48 MB)
-
MP-IVM in the tibia of the CX3CR-GFP mouse model in which macrophages (MCs, white arrows in Fig. 2A) and dendritic cells (bmDCs, red arrows in Fig. 2A) are identified by a combination of fluorescence and cell shape.
- Video 3. Visualization of CFSE stained, i.v. transplanted L-S-K+ progenitor cells that homed close to the endosteum in the tibia (AVI, 741 KB)
-
Fluorescently labeled cells inside the bone cavity were analyzed by MP-IVM 16–40 hours post transplantation as described in Fig. 1 and Document 1.
- Video 4. Visualization of a CFSE stained, i.v. transplanted L-S+K+ early progenitor cell that homed close to the endosteum in the tibia (AVI, 609 KB)
-
Fluorescently labeled cells inside the bone cavity were analyzed by MP-IVM 16–40 hours post transplantation as described in Fig. 1 and Document 1.
- Video 5. Visualization of a CFSE stained, i.v. transplanted aged (20 month old) L-S+K+ progenitor cells that homed close to the endosteum in the tibia (AVI, 595 KB)
-
Fluorescently labeled cells inside the bone cavity were analyzed by MP-IVM 16–40 hours post transplantation as described in Fig. 1 and Document 1.
|
|
