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>