明日はここからよ。うふふふふ。
Cell size measurement
Marrowspheres from 4-week-old mice, collected after 5 days in the fresh medium, were dissociated into single cells.
感想は全部読んでからにしましょ。
Dissociated cells were stained with propidium iodide filtered through a 40 mm pore filter and measured on a DAKO Galaxy (DAKO) using FloMax software.
あら、失礼。私も小保方さんと性格が似てるのよ。うふふふふ。
Microbeads of predefined sizes (Size Calibration Standards Kit; Bangs Laboratories, Inc.) were re-suspended in isotonic phosphate saline (pH 7.2) and used as a standard for whichto compare size of cells contained in spheres using cytofluorimetric analysis.
そういう時は先を読むとヒントがあったりなかったり、余計分からなくなったり。。。
Both cells and beads were analyzed using the same instrument setting (forward scatter, representing cell and bead size, and side scatter, representing cellular granularity).
ワトソンさん。そのギャグはホームズさんに飛ばしてくださらない。
はい、これでおしまいよ。
Cell size was calculated on a curve employing bead size on the x-axis and forward scatter values on the axis.
ドットプロット解析図なんでしょ? 僕の理解だと
a curve employing bead size on the x-axis ってMicrobeads of predefined sizesの結果なんでしょ。
全部同じものだからカーブにならないんじゃないの。一か所に集中するはずだけど。そこに
本物のスフィア細胞の単離したものを飛ばすんでしょ。どのあたりに分布するかを見て大きさを
比較する。何か間違って理解してるね。
忘却とは忘れ去ることなり。
Each slide was fixed with 4% paraformaldehyde (Wako) for 15 min at room temperature, washed with PBS, incubated with SuperBlock blocking buffer in PBS (P74370; Takara) for 30 min to block nonspecific reactions, and incubated with anti-c-kit rat monoclonal antibody (sc-19619; Santa Cruz Biotechnology, Inc), anti-Sca-1 rat monoclonal antibody (ab25031; Abcam), or anti-E-cadherin rat monoclonal antibody (ab11512; Abcam) overnight at 48C.
�B
専門的な手順を語ってるんでしょ。
After washing with PBS, the cells were incubated with goat anti-rat IgG Texas Red-conjugated antibodies (112-076-062;Jackson ImmunoResearch) and goat anti-rat IgG Fluorescein-conjugated antibodies (112-096-062; Jackson Immuno-Research) for 30 min at room temperature.
ここは彼女の専門ですからね。
Stage-specific embryonic antigen-1 and alkaline phosphatase staining was performed using the ES cell detection Kit (Millipore) in accordance with the manufacturer’s protocol.
少しは、興味が出てきましたか?それとも今日も釣りなの?
In negative controls, the primary antibody was replaced with IgG-negative controls of the same isotype to ensure specificity.
はいはい、詰まんないことやってないで前へ進んでください。
The spheres were collected at 5 days and dissociated into single cells, and placed in DMEM supplemented 20% fetal calf serum (FCS).
The spheres were collected at 5 days and dissociated into single cells, and placed in DMEM supplemented 20% fetal calf serum (FCS).の
single cellsとAfter 7–14 days, muscle cells were stained のmuscle cells とどういう関係にあるのかね?そもそも、The spheres ってどの組織から
得られたものなのかね?
