Fluorescence microscopy B cell data set

Fluorescence microscopy data set of B cells with corresponding ground truth. The images were captured with an Zeiss AxioSscan.Z1.<br>Sample Preparation:Naive murine B cells from C57Bl/6 mice were isolated from the spleen by negative selection. The cells<br>were activated with lipopolysaccharide (LPS, 10 <i>µ</i>g/ml) for 72 h in RPMI1640 medium supplemented with<br>fetal calf serum (FCS)(10%), L-glutamine (2 mM), Pyruvate (1 mM), Penicillin (50 U/ml), Streptomycin<br>(50 <i>µ</i>g/ml) and ?-Mercaptoethanol (50 <i>µ</i>M). Teflon-coated microscope slides with 8 wells each were<br>coated with aBCR (10 <i>µ</i>g/ml rat anti BCR monoclonal) [1] or αCD19 (rat anti CD19<br>monoclonal) [3]. 2x10^4 B cells were seeded on the slides and incubated for 45min in a<br>humidified incubator (5% CO2 atmosphere in RPMI1640 supplemented as described above but without<br>FCS). On the control sample, B cells were treated with Cytochalasin D [2], a mycotoxin<br>that inhibits actin polymerization. Cell were fixated in phosphate buffered saline (PBS) containing 4%<br>para-formaldehyde which also stops cell spreading. Fixed cells were washed and permeabilized in PBS with<br>0.1% Triton X-100. Phalloidin-Rhodamin (Molecular Probes) specifically stained the F-actin intracellularly<br>and DAPI (Roth) stains DNA in the nuclei. Slides were mounted in MOWIOL(Roth) [4].<br>References:[1] Cambier, J. C., Heusser, C. H., and Julius, M. H. (1986). Abortive activation of B lymphocytes by<br>monoclonal anti-immunoglobulin antibodies. Journal of immunology (Baltimore, Md. : 1950) 136,<br>3140–6<br>[2] Cooper, J. A. (1987). Effects of cytochalasin and phalloidin on actin. The Journal of Cell Biology 105,<br>1473–1478. doi:10.1083/jcb.105.4.1473<br>[3] Krop, I., Shaffer, A. L., Fearon, D. T., and Schlissel, M. S. (1996). The signaling activity of murine CD19<br>is regulated during cell development. Journal of immunology (Baltimore, Md. : 1950) 157, 48–56<br>[4] Wiesmann, V., Reimer, D., Franz, D., Hüttmayer, H., Mielenz, D., and Wittenberg, T. (2015). Automated<br>high-throughput analysis of B cell spreading on immobilized antibodies with whole slide imaging.<br>Current Directions in Biomedical Engineering 1, 224–227. doi:10.1515/cdbme-2015-0056<br>

本数据集为带有对应真值标注（ground truth）的B细胞荧光显微镜数据集，图像通过蔡司AxioSscan.Z1显微镜采集获得。

样本制备：
从C57Bl/6小鼠脾脏中通过阴性分选法分离得到初始态小鼠B细胞。将细胞置于添加了10%胎牛血清（fetal calf serum, FCS）、2mM L-谷氨酰胺、1mM丙酮酸钠、50U/ml青霉素、50μg/ml链霉素以及50μM β-巯基乙醇的RPMI1640培养基中，使用10μg/ml脂多糖（lipopolysaccharide, LPS）刺激活化72小时。

将每孔带有特氟龙涂层的8孔显微镜载玻片分别包被10μg/ml大鼠抗B细胞受体（B cell receptor, BCR）单克隆抗体[1]或抗CD19大鼠单克隆抗体[3]。随后将2×10^4个B细胞接种至载玻片上，于加湿培养箱中孵育45分钟，培养条件为含5%CO₂的RPMI1640培养基（配方同前，但不含FCS）。

对照组样本中的B细胞经细胞松弛素D（Cytochalasin D）处理，该物质为一种可抑制肌动蛋白聚合的真菌毒素。将细胞置于含4%多聚甲醛的磷酸盐缓冲液（phosphate buffered saline, PBS）中固定，该溶液同时可终止细胞铺展过程。固定后的细胞经洗涤后，使用含0.1%曲拉通X-100（Triton X-100）的PBS进行透化处理。罗丹明标记的鬼笔环肽（Phalloidin-Rhodamin，Molecular Probes品牌）可特异性对细胞内的纤维状肌动蛋白（F-actin）进行染色，而DAPI（Roth品牌）则可对细胞核内的DNA进行染色。最终使用MOWIOL试剂（Roth品牌）对载玻片进行封片处理。

参考文献：
[1] Cambier, J. C., Heusser, C. H., Julius, M. H. (1986). 单克隆抗免疫球蛋白抗体诱导的B淋巴细胞流产型活化. 《免疫学杂志（Journal of immunology）》（美国马里兰州巴尔的摩，1950年创刊），第136卷，第3140-3146页
[2] Cooper, J. A. (1987). 细胞松弛素与鬼笔环肽对肌动蛋白的影响. 《细胞生物学杂志（The Journal of Cell Biology）》，第105卷，第1473-1478页，doi:10.1083/jcb.105.4.1473
[3] Krop, I., Shaffer, A. L., Fearon, D. T., Schlissel, M. S. (1996). 小鼠CD19的信号活性在细胞发育过程中受到调控. 《免疫学杂志（Journal of immunology）》（美国马里兰州巴尔的摩，1950年创刊），第157卷，第48-56页
[4] Wiesmann, V., Reimer, D., Franz, D., Hüttmayer, H., Mielenz, D., Wittenberg, T. (2015). 基于全玻片成像的B细胞在固定化抗体上铺展的自动化高通量分析. 《生物医学工程新进展（Current Directions in Biomedical Engineering）》，第1卷，第224-227页，doi:10.1515/cdbme-2015-0056