Journal: Diabetes

Article Title: Measurements of CD34+/CD45-dim Stem Cells Predict Healing of Diabetic Neuropathic Wounds

doi: 10.2337/db15-0517

Figure Lengend Snippet: HIF ratio comparison between Western blotting and flow cytometry techniques. Left: Representative SPC Western blots (entire blots are shown) from three cell lysates (A, B, and C). HIF-1 was monitored in the scanner's 700-nm channel, and HIF-2 and HIF-3 were monitored in the 800-nm channel. Anticipated molecular weight for HIF-1 was ∼93 kDa; according to the antibody manufacturer, HIF-2 typically exhibits two isoforms at 120 and 96 kDa; and HIF-3 molecular weight was 69 kDa. Right: Linear regression comparison between HIF ratios calculated from flow cytometry versus Western blot data. These comparisons were made using cells from 11 study subjects.

Article Snippet: For immunohistochemistry, the following antibodies were used: fluorescein isothiocyanate–conjugated mouse anti-human CD34 (Invitrogen, Grand Island, NY), RPE-conjugated mouse anti-human CD45 (Santa Cruz Biotechnology, Dallas, TX), allophycocyanin-conjugated mouse anti-human CD133 (Miltenyi Biotec), RPE-conjugated goat anti-human HIF-1 (R&D Systems), and A647-conjugated rabbit anti-human HIF-2 and RPE-conjugated rabbit anti-human HIF-3 (Bioss, Inc.).

Techniques: Western Blot, Flow Cytometry, Molecular Weight

Journal: Diabetes

Article Title: Measurements of CD34+/CD45-dim Stem Cells Predict Healing of Diabetic Neuropathic Wounds

doi: 10.2337/db15-0517

Figure Lengend Snippet: Flow cytometry blood SPC analysis for HIF proteins. After forward (FSC-H) and side scatter (SSC-H) interrogation, the P1 zone was selected and cells positive for CD34 and negative for CD45 (defined based on the FMO Control Test [box P3] were analyzed for content of HIF proteins). Histograms show fluorescence for cells incubated with antibodies to HIF-1, HIF-2, and HIF-3, as well as isotype control antibodies. FITC, fluorescein isothiocyanate; PE, phycoerythrin.

Article Snippet: For immunohistochemistry, the following antibodies were used: fluorescein isothiocyanate–conjugated mouse anti-human CD34 (Invitrogen, Grand Island, NY), RPE-conjugated mouse anti-human CD45 (Santa Cruz Biotechnology, Dallas, TX), allophycocyanin-conjugated mouse anti-human CD133 (Miltenyi Biotec), RPE-conjugated goat anti-human HIF-1 (R&D Systems), and A647-conjugated rabbit anti-human HIF-2 and RPE-conjugated rabbit anti-human HIF-3 (Bioss, Inc.).

Techniques: Flow Cytometry, Fluorescence, Incubation

Journal: Diabetes

Article Title: Measurements of CD34+/CD45-dim Stem Cells Predict Healing of Diabetic Neuropathic Wounds

doi: 10.2337/db15-0517

Figure Lengend Snippet: Fluorescence values for HIF proteins in blood SPCs. Data show median fluorescence and 25th and 75th percentiles for CD34+/CD45-dim cell expression of HIF-1, HIF-2, and HIF-3 and the HIF ratio calculated as the median fluorescence of HIF-1 plus HIF-2 divided by HIF-3. The sample number for each value is as shown in Fig. 1; there are no statistically significant differences between groups. Strt, start; Wk, week.

Article Snippet: For immunohistochemistry, the following antibodies were used: fluorescein isothiocyanate–conjugated mouse anti-human CD34 (Invitrogen, Grand Island, NY), RPE-conjugated mouse anti-human CD45 (Santa Cruz Biotechnology, Dallas, TX), allophycocyanin-conjugated mouse anti-human CD133 (Miltenyi Biotec), RPE-conjugated goat anti-human HIF-1 (R&D Systems), and A647-conjugated rabbit anti-human HIF-2 and RPE-conjugated rabbit anti-human HIF-3 (Bioss, Inc.).

Techniques: Fluorescence, Expressing

Journal: Diabetes

Article Title: Measurements of CD34+/CD45-dim Stem Cells Predict Healing of Diabetic Neuropathic Wounds

doi: 10.2337/db15-0517

Figure Lengend Snippet: Representative confocal microscope images of wound margin tissue from two patients at week 1 follow-up visit from a patient who healed (A) and from a patient who did not heal within 16 weeks (B). One set of tissue sections was stained with antibodies to CD34 as well as HIF-1 and HIF-2 and another with CD34 and HIF-3; merge images depict overlap between respective protein staining. The table beneath the images shows the quantitative analysis for the sections as median CD34+ cells/image and percent of cells exhibiting fluorescence for HIF-1, HIF-2, and HIF-3. The orientation for all samples was epidermis toward the top of the image.

Article Snippet: For immunohistochemistry, the following antibodies were used: fluorescein isothiocyanate–conjugated mouse anti-human CD34 (Invitrogen, Grand Island, NY), RPE-conjugated mouse anti-human CD45 (Santa Cruz Biotechnology, Dallas, TX), allophycocyanin-conjugated mouse anti-human CD133 (Miltenyi Biotec), RPE-conjugated goat anti-human HIF-1 (R&D Systems), and A647-conjugated rabbit anti-human HIF-2 and RPE-conjugated rabbit anti-human HIF-3 (Bioss, Inc.).

Techniques: Microscopy, Staining, Fluorescence