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