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Blood, Vol. 111, Issue 8, 4220-4232, April 15, 2008
NSOM/QD-based nanoscale immunofluorescence imaging of antigen-specific T-cell receptor responses during an in vivo clonal V 2V 2 T-cell expansion
Blood Chen et al.
111: 4220
Supplemental materials for: Chen et al
MATERIALS AND METHODS Control experiments for avoiding non-specific antibody staining
To ensure the antibody specific staining of V 2+, V 2+, V 3+, V17+ or V5+ T cells, following controls were always included. (1) PBL were incubated with IgG isotype-matched control antibodies followed by QD-conjugated Ab or streptavidin, or FITC− or PE−, APC−, or Pacific blue-conjugated Ab. Flow cytometry and confocol microscopy analyses showed an absence of detectable non-specific staining in the setting of the control experiments. (2) PBL were incubated with QD-conjugated Ab, and then magnetically-labeled by a 30-min incubation with goat anti-mouse IgG microbeads as described above, and selected by the Mini MACS Separator. There was no detectable positive staining of these cells, suggesting a lack of non-specific biding to PBL by QD-conjugated Ab or streptavidin In the initial experiments, CD3, CD4 or CD8 antibody staining of PBL followed by QD-conjugated Ab was also included as positive controls to ensure that the whole staining procedure worked.
Files in this Data Supplement:
- Figure S1. γδ TCR on unstimulated T cells were distributed differently from their αβ TCR counterparts, with single individual γδ TCR dominant on Vδ2+ T cells and clustered αβ TCR complex more evident on various Vβ-expressing T cells (JPG, 155 KB)
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(A) show topographic (left), fluorescence (middle) and merged topographic-fluorescence (right) NSOM images of  TCR on V5+ T cells. (B) show topographic (left) and fluorescence (right) NSOM images of TCR on V2+ T cells. Note the fluorescent V5+ T cell in Fig. S2a was one of the three scanned cells as shown in the two lower-magnification graphs (25 µm × 12 µm) inserted in the lower left (topographic) and lower right (fluorescence) bottom, respectively. (C) NSOM topographic and fluorescence images of V3.1+ (c1) and V17+ (c2) T cells. Left panel: lower-magnification NSOM topographic and fluorescence graphs (25 µm × 25 µm); Middle and right panels: high-magnification NSOM topographic (middle panel) and corresponding fluorescence (right panel) images (9 µm × 9 µm in c1; 10 µm × 10 µm in c2) of the cell marked by the open squares in left panel. Scan size: (A) 8.3 µm × 8.3 µm; (B) 14 µm × 8 µm. Resolution: (left panel in C) 300pixel × 300pixel; (A, B, and middle and right panel in C) 500pixel × 500pixel. Integration time: (A) 15 ms; (B, C) 20 ms.
- Figure S2. Clonally-expanded Vγ2Vδ2 T cells examined on days 4 and 7 after the HMBPP/IL-2 treatment exhibited TCR aggregates or capped dots under confocal microscopy (JPG, 358 KB)
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Shown are representative confocal microscopic (both fluorescence and DIC) images of V2+ (left lane) or V2+ (right lane) T cells collected from peripheral blood of three monkeys on day 0, day 4, day 7, day11, week 3, week 12 after the HMBPP/IL-2 treatment. Imaging data (boxed by black frames) were marked with individual time points of D0, D4, D7, D11, W3, and W12, respectively, on the upper left corner. In general, the first panel shows low-magnification images with many cells, while the second and third panels show the center and surface parts of the same cells in higher magnification, respectively. In the images, Qdot655-labeled anti-V2 / anti-V2 is red, and the gray images are corresponding DIC. The relevant flow cytometric data are shown in the Fig. 4a of the text. Almost all expanded T cells after HMBPP/IL-2 treatment were V2V2 T cells (cells co-expressing both V2 and V2). For each time point, at least 100 V2+ or V2+ T cells were analyzed for the presence of TCR aggregates or capped dots. On days 4 and 7 after the HMBPP/IL-2 treatment, all the V2V2 T cells examined by the confocal microscopy exhibited TCR aggregates or capped dots, whereas > 50% of V2V2 T cells examined on day 11 had those TCR aggregates. Unstimulated V2V2 T cells in PBL collected on day 0 from the monkeys before the HMBPP treatment did not have any apparent aggregates or capped dots.
- Figure S3. Clonally-expanded Vγ2Vδ2 T cells examined on days 4 and 7 after the Picostim/IL-2 treatment exhibited TCR aggregates or capped dots under confocal microscopy (JPG, 434 KB)
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Shown are representative confocal microscopic (both fluorescence and DIC) images of V2+ (left lane) or V2+ (right lane) T cells collected from peripheral bloods of 3 monkeys on day 0, day 4, day 7, week2, week 5, week 12 after the Picostim/IL-2 treatment. Imaging data (boxed by black frames) were marked with individual time points of D0, D4, D7, W2, W5, and W12, respectively, on the upper left corner. The first panel generally shows low-magnification images with many cells, while the second and third panels show the center and surface parts of the same cells in higher magnification, respectively. In the images, Qdot655-labeled anti-V2 / anti-V2 is red, and the gray images are corresponding DIC. The relevant flow cytometric data are shown in the Fig. 4B of the text. For each time point, at least 100 V2+ or V2+ T cells were analyzed for the presence of TCR aggregates or capped dots. On days 4 and 7 after the Picostim/IL-2 treatment, all the V2V2 T cells examined by the confocal microscopy exhibited TCR aggregates or capped dots, whereas > 50% of V2V2 T cells examined at week 2 had those TCR aggregates. No apparent TCR aggregates were seen in unstimulated V2V2 T cells in PBL collected before Picostim treatment from the monkeys.
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