Additional file 2: of Comparison of pre-processing methods for multiplex bead-based immunoassays

Supplementary table. Detailed results for the evaluation criteria skewness, tail length and coefficient of variation. (PDF 54 kb

Additional file 1: of Comparison of pre-processing methods for multiplex bead-based immunoassays

Supplementary figures. All results for the QQ-, Mean-SD, Bland-Altman and Volcano plots. (PDF 5133 kb

Additional file 3: of Comparison of pre-processing methods for multiplex bead-based immunoassays

Supplementary material. Datasets, which support the conclusions of this article. (ZIP 58 kb

Expression of neuronal and glial markers in teratomas grown in 129Sv and SCID/beige mice after injection of ES and differentiated cells.

<p>Teratomas were obtained after injection of ES cells or differentiated (diff.) cells into syngeneic 129Sv mice and immunodeficient SCID/beige mice. Histological sections were stained by immunohistochemistry for the neuronal marker NeuN and the glial marker GFAP. The presence of the markers is indicated semi quantitatively in four categories: − negative, + single positive cells, ++ groups of positive cells, +++ confluent groups of positive cells. Five representative teratomas per group were analyzed.</p>1<p>2 of 5 teratomas were negative for this marker.</p>2<p>3 of 5 teratomas were negative for this marker.</p

Some of the ES cell-derived neuronal colonies still contain at day 14 OCT3/4 and Ki67-positive cells.

<p>(A) After 14 days of differentiation culture few colonies (less than 5%), as exemplified here, still contain OCT3/4-positive cell clusters as detected by immunofluorescence staining using a specific mAb (magnification 40×). (B) The same colony was stained by an anti-Ki67 mAb to detect proliferating cells. (C) The merged staining indicates that Ki67-positive proliferating cells exist which are not OCT3/4-positive stem cells. (D) In parallel, cells of the neuronal differentiation culture were stained at day 14 by anti-Ki67 and anti-Tuj1 mAb. The merged staining (magnification 20×) indicates that the vast majority of the cells are Tuj1-positive neuronal cells. Few Ki67-positive proliferating cells are negative for the neuronal cell marker.</p

Tumor formation after subcutaneous inoculation of various numbers of ES cells or <i>in vitro</i> differentiated cells in 129Sv mice.

<p>The indicated number of ES cells and cells differentiated <i>in vitro</i> for 14 days were injected subcutaneously into the flank of 129Sv mice. The percentage and number of animals is indicated in which tumors were found during autopsy at the side of injection before day 100 after injection.</p

Tumor formation after subcutaneous inoculation of ES or <i>in vitro</i> differentiated cells into various hosts.

<p>ES cells and cells differentiated <i>in vitro</i> for 14 days were injected subcutaneously into the flank of syngeneic or allogeneic mice or xenogeneic rats (1×10⁶ cells in PBS/animal). Some recipients received an immunosuppressive treatment with CsA (10 mg/kg/day). The percentage and number of animals in which tumors were found during autopsy or in which tumors were palpable (at least during 3 consecutive observations) at the side of injection before day 100 after injection is indicated.</p>1<p>in 2 mice (8%) a tumor regression was observed before day 100.</p>2<p>in 2 mice (9%) a tumor regression was observed before day 100.</p>3<p>in 1 mouse (7%) a tumor regression was observed before day 100.</p>4<p>in 2 mice (11%) a tumor regression was observed before day 100.</p>5<p>in 8 female mice (47%) a tumor regression was observed before day 100.</p>6<p>in 2 rats (11%) a tumor regression was observed before day 100.</p

Lysis of ES and <i>in vitro</i> differentiated cells by splenocytes derived from rats 6 weeks after intracerebral grafting of <i>in vitro</i> differentiated cells.

<p>Mean of specific lysis and SD of triplicates of ES cells (A, C, E, G) or <i>in vitro</i> differentiated cells (B, D, F) at three effector∶target (E∶T) ratios. Effector cells were lymphocytes obtained by density gradient centrifugation on Biocoll from spleens of grafted (no. 113–117) or naïve (co1 and co2) DA rats (A, B), grafted (no. 118–122) or naïve (co1 and co2) LEW.1N rats (C, D), and grafted Wistar rats (no. 38, 39, 48, 49, 51, 52, 53, 54, 56, 57). The individual rats are indicated by symbols. (G) Mean of specific lysis and SD of triplicates of ES cells by lymphokine-activated killer (LAK) cells derived from splenocytes of Wistar rats (no. 38, 48, 49, 51, 52, 54, 56) after culture for 4 days in the presence of 100 U/ml IL-2.</p

Analysis of effects of NK cells on the rejection of ES cells in vivo.

<p>(A) 1×10⁶ ES cells were injected subcutaneously at day 0 into 15 T and B cell deficient SCID mice which have functional NK cells. The tumor size was recorded every second day until day 100 using linear calipers. The growth of tumors in individual mice is shown. Tumor growth in female hosts is indicated by red lines and in male hosts by blue lines. In one animal a tumor regression was observed. (B) The treatment scheme of rats receiving an NK cells depleting antibody (anti-NKR-P1A) or an isotype control is shown. (C) The mean proportion plus SD of NK cells in the blood of male LOU rats receiving the anti-NKR-P1A mAb (n = 8) or the isotype control (n = 6) is shown. Blood cells were stained with the respective mAb and analyzed by flow cytometry after lysis of erythrocytes. (D) The proportion of the male rats in which tumors were found at autopsy is indicated. The size of the tumors was 12 mm³ and 16 mm³, respectively. Both were palpable for more than 30 days before autopsy.</p

Immunohistochemical staining identifies neuronal and glial cells in teratomas derived from ES and <i>in vitro</i> differentiated cells.

<p>The tumors were grown in SCID/beige mice after injection of ES cells (A, C) or after injection of <i>in vitro</i> differentiated cells (B, D). The teratomas were stained by immunohistochemistry for the neuronal marker NeuN (A, B) and the glial marker GFAP (C, D) which is mainly found in astrocytes. In the sections shown here, groups of cells positive for NeuN and confluent groups of cells positive for GFAP were found.</p