|
|
Blood, Vol. 110, Issue 4, 1215-1224, August 15, 2007
Splenic CD19–CD35+B220+ cells function as an inducer of follicular dendritic cell network formation
Blood Murakami et al.
110: 1215
Supplemental materials for Murakami et al, Vol 110, Issue 4, 1215-1224
Files in this Data Supplement:
- Figure S1. Ethanol fixation does not alter the intensity of the fluorescent dye PKH26 (JPG, 9.52 KB)
-
To confirm if ethanol fixation diminishes the intensity of PKH26 fluorescence, WEHI3 cells were stained with PKH26 and fixed with 70% ethanol. The intensity of PKH26 fluorescence was assessed by fluorescence activated cell sorting (FACS) with a FACSCalibur. The red line indicates no staining; the black line, PKH26-labeled cells without ethanol fixation; the green line, PKH26-labeled cells with ethanol fixation.
- Figure S2. CD35+B220+ cells having a ruffled cell membrane (JPG, 14.9 KB)
-
(A) Splenic CD35+B220+ cells isolated from C57BL/6J-Jcl mice were placed in a glass-bottomed dish. DIC time-lapse images (2 minutes, 150 seconds each) show dynamic changes in the shape of CD35+B220+ cells. DIC images were obtained with a DeltaVision RT system, and a representative of three independent experiments is shown. (B) CD35+B220+ cells isolated from GFP-Tg mice, having a ruffled cell membrane. Isolated CD35+B220+ cells were cultured in a glass-bottomed dish for 24 hours. The cells were air-dried and fixed with 4% buffered paraformaldehyde. Images were taken and analyzed with a DeltaVision RT system and an objective magnification of 60×. Details on photography are in “Materials and methods, Observation and photography.”
- Figure S3. CD35+B220+ cells fuse with stromal cells to form double-nuclear cells (JPG, 21.8 KB)
-
CD35+B220+ cells were cultured with or without CD45−CD35− splenic stromal cells for 5 days. Double-nuclear cells found in the co-culture system (A) or monoculture of CD35+B220+ cells (B) were examined with scanning electron microscopy (SEM) at a magnification of 1500×. Details on SEM analyses are in “Materials and methods.” Scale bars are shown.
- Figure S4. CD35+B220+ cells enhance formation of splenic CD35+ reticulum upon adoptive transfer (JPG, 11.3 KB)
-
CD35+B220+ cells or ethanol-fixed CD35+B220+ cells were injected intravenously (105 cells/body) into naïve C57BL/6J-Jcl mice. On days 5 and 8 after injection, frozen sections of spleen were prepared and stained with biotin-conjugated anti-CD35 mAb, followed by diaminobenzidine (DAB) visualization. The number of CD35+ clusters in the splenic sections was counted under a light microscope. Each group consisted of 5 mice and at least 10 different parts of each spleen were counted. P<.05.
|
|
