Computational protocol: Geometrically Controlled Asymmetric Division of CD4+ T Cells Studied by Immunological Synapse Arrays

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Protocol publication

[…] CD4+ T cells suspended in RPMI 1640 medium (Life Technologies) supplemented with 10% of FBS (Life Technologies), 100 U/ml of penicillin, 100 mg/ml of streptomycin (Life Technologies), and 0.1% of beta-mercaptoethanol (Sigma) were seeded on the ISAs with ∼ 106 cells/ml of density. ISAs containing T cells were then incubated for 32 h in a tissue culture incubator maintaining 5% CO2 and 37°C, fixed with 4% paraformaldehyde for 15 min at room temperature, washed with PBS, and stained with fluorescent-labeled antibodies. A modified Zeiss Axio Observer.Z1 epifluorescence microscope with a 40X objective lens (Plan-Neofluar, NA = 1.30) and a Roper Scientific CoolSnap HQ CCD camera was used for imaging. XBO 75 W/2 Xenon lamp (75 W, Osram) and DAPI (EX. 365, BS 395, EM BP 445/50), eGFP (EX BP 470/40, BS 495, EM BP 525/50), Cy3 (EX BP 550/25, BS 570, EM BP 605/70), Cy5 (EX BP 620/60, BS 660, EM BP 770/75) excitation/emission filter sets were used for illumination and fluorescence imaging. The microscope was automatically controlled by Axiovision 4.6, (Carl Zeiss) and acquired images were analyzed and processed with Metamorph (Universal Imaging, Molecular Devices) or ImageJ (NIH). Fluorescence images of fixed and stained T cells on the ISAs were acquired by optical sectioning (8 individual planes, 1 μm apart) and analyzed by integration through z-sections. […]

Pipeline specifications

Software tools MetaMorph, ImageJ
Applications SPIM, Microscopic phenotype analysis