Retrobulbar administration of TPCA-1 causes no obvious toxicity to blood cells.

(A–F) Flow cytometric analyses of blood cell population. Single cell suspension was prepared from spleen, thymus and bone marrow of the mice at 7 days after laser-injury followed immediately by a single retrobulbar injection of 56 μg TPCA-1 in 20 μL vehicle solution (PBS/20% DMSO) or control vehicle only in 20 μL. The representative plots are shown and numerical values represent the average (±SD, n>5). Statistics were performed using ProStat 5.5 program, and showing no significant difference in all the groups. P≤0.05 is considered as significant. (A, B) The CD4⁺ and CD8a⁺ T cells from the TCRβ-gated populations in spleen (A) and thymus (B) were calculated and plotted. (C, D) Spleen (C) and bone marrow (D) cells were isolated from TPCA-1 treated (+) and untreated (−) mice and stained with anti-B220, anti-IgM, and anti-IgD antibodies. Data in (C) show the percentile of mature (IgD^highIgM^low) and immature (IgD^lowIgM^high) B cells in the B220-gated splenocytes, and data in (D) show the percentages of mature recirculating (R2, IgD^highIgM^int), transitional immature (R3, IgM^highIgD^int), immature (R4, IgM^highIgD⁻) and precursor (R5, IgM⁻IgD⁻) B cell populations in the B220-gated bone marrow cells. (E) Spleen cells were isolated from TPCA-1 treated (+) and untreated (−) mice and stained with anti-CD11b, anti-Ly6C and anti-Ly6G antibodies. The numerical values are shown as the average of neutrophil (Ly6G⁺/Ly6C^int), monocyte (Ly6G⁻/Ly6C⁺) and mature macrophage (Ly6G⁻/Ly6C⁻) from the CD11b gate in the spleens. (F) Spleen cells were stained with 7AAD to detect dead cells. The numerical values are shown as the average of the CD11b⁺ 7AAD⁺ populations in splenic lymphoid cells. (G) Hematoxylin and eosin (H&E) histological staining was performed to evaluate toxicity to liver and spleen at 7 days after treatment. The scale bar represents 50 μm in length. (H) Relative cell viability of HEK293T cells (left panel) and ARPE-19 cells (right panel) was shown at 12 hours after being cultured with 50 μM TPCA-1 or 10 ng/mL TNFα or combination of both. The cells were pretreated with 50 μM TPCA-1 for 30 minutes prior to addition of 10 ng/mL TNFα for combination treatment. Cell viability was measured using MTT assay. Data are represented as the mean±SD from three independent experiments. Asterisk indicates Two-tail T-test: *** p<0.005.