Confocal microcopy was preformed as described (Chang et al., 2011 (link); Lin et al., 2015 (link)). Using transmitted light morphology, tubulin bridge staining, and DNA staining, specific identification of telophase or cytokinetic sibling pairs with exclusion of any spurious neighboring-but-unrelated cell pairs is achieved (Lin et al., 2015 (link)). For fixed cell imaging, cells were adhered to poly-l-lysine (Sigma) coated coverslips (#1.5 Thermo Fisher) and treated with freshly prepared 3.7 % PFA (Sigma) diluted in cell culture media (pH 6.8) in order to preserve mitochondrial morphology and MitoTracker Red FM fluorescence intensity. Following brief treatment with 0.1% triton cells were stained with antibody against α-Tubulin (clone YOL1/34 Abcam; 1:300) and then anti-rabbit antibody conjugated to AlexaFluor568 (Thermo Fisher) in a PBS solution containing 0.01 % saponin and 0.25 % fish skin gelatin (Sigma). For live cell imaging, cells were transferred to poly-l-lysine coated 8-chambered coverglass wells (#1 LabTek) in warm cell culture media and allowed to adhere briefly before imaging. Images were acquired on inverted confocal microscopes (Zeiss LSM710 or Nikon Ti Eclipse) and processed using ImageJ (v.1.46r) or Fiji (v.2.0) software. Total fluorescence in defined regions of single cells was quantitated using the integrated density function in ImageJ.
Coated coverslips
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
Coated coverslips are a type of lab equipment used to support and protect biological samples during microscopic observation. They feature a specialized coating that enhances the attachment and growth of cells or other specimens. The core function of coated coverslips is to provide a suitable substrate for the examination and analysis of samples under a microscope.
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Lab products found in correlation
Poly l lysine (pll), by Merck Group (3 mentions)
24 well plate, by Jet Biofil (2 mentions)
Hek293 cells, by Merck Group (2 mentions)
Phosphate buffered saline (pbs), by Merck Group (2 mentions)
Fish skin gelatin, by Merck Group (1 mentions)
Lsm710, by Nikon (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Eclipse ti, by Nikon (1 mentions)
Dapi fluoromount, by Southern Biotech (1 mentions)
Poly d lysine, by Merck Group (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Eclipse ti2 fluorescence microscope, by Nikon (1 mentions)
Ds ri2 digital camera, by Nikon (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Coliv, by Merck Group (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Microscope slides, by Vector Laboratories (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
7 protocols using coated coverslips
1
Mitochondrial Morphology Imaging and Analysis
2
Quantifying p-eIF2α in HEK cells
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HEK cells grown on poly-D-lysine (Sigma Aldrich)-coated cover slips (Thermo Scientific) were treated for 24 h with geneticin in F10 medium with 15 μg/ml saponin, or with arsenite for 1 h (positive control). Cells were then fixed with 4% paraformaldehyde and methanol and incubated with blocking solution (1 × PBS with 1% BSA and 0.5% saponin) for 1 h at RT. Immunostaining used a rabbit polyclonal antibody against p-eIF2α (1 : 250) and a secondary goat anti-rabbit antibody conjugated with Texas Red (1 : 250) diluted in blocking solution. Cover slips were mounted on glass slides (VWR) using Dapi fluoromount (Southern Biotech, Birmingham, AL, USA), and cells were imaged using a Lyca Sp2 confocal microscope and a 63 × objective. p-eIF2α and nuclear signals were quantified using Imaris software (Bitplane, Belfast, UK) and the dots-per-cell ratio was calculated.
3
Memantine and Aspirin Effects on NR2B Expression
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SH-SY5Y cells were grown on coated coverslips (Thermo Fisher Scientific, Waltham, MA, USA) and differentiated into neuron-like cells. The cells were treated with 0.25 μg/mL memantine for 6 h and then treated with 40 μg/mL aspirin for 8 h. Thereafter, the cells were stained with primary antibody against NR2B (1:900; Invitrogen, Carlsbad, CA, USA) for 2 h at room temperature (RT), and then with the donkey anti-rabbit IgG secondary antibody (1:400, conjugated with Alexa 555; Molecular Probes Inc., Eugene, OR, USA) together with Hoechst 33342 (1:1000; Molecular Probes Inc., USA) for 1 h 30 min at RT. After washing with phosphate-buffered saline (PBS), the cells were mounted (ProLong Gold anti-fade reagent; Molecular Probes Inc., USA) and visualized under a Nikon Eclipse Ti2 fluorescence microscope (Nikon, Tokyo, Japan). Cell images were taken with a DS-Ri2 digital camera (Nikon, Japan).
4
Directed Differentiation of hESC-RPE Cells
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When the amount of cells was sufficient, the hESC-RPE cells were dissociated with 1× Trypsin–EDTA again and plated on top of Ormocomp-treated (Micro Resist Technology GmbH, Germany) [29 (link)], ColIV (Sigma-Aldrich)-coated coverslips (7 mm in diameter; Thermo Fisher Scientific, Inc., Leicestershire, UK) at a density of 105 cells/cm2. Cells were cultured for a period of 9 ± 1, 16 ± 1, 28 ± 1, or 35 ± 1 days in RPEbasic medium, during which the medium was replenished thrice a week.
5
Tau4R-GFP Transduction for Imaging
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Polyclonal Tau4R-GFP cells were plated at 2 × 105 per well in 24-well plates. Cells were transduced the next day, using 40 μl of 10% clarified brain homogenate combined with Opti-MEM to a final volume of 50 μl. A further 48 μl of Opti-MEM and 2 μl of Lipofectamine-2000 (Invitrogen) was added to the previous Opti-MEM mixture to a total volume of 100 μl and incubated for 20 min. The liposome mixture was applied to the cells for 18 hr, and cells were then washed with PBS, trypsinized, and re-plated on coated coverslips (ThermoFisher) for imaging and analysis.
For PTA-precipitated brain homogenate, 1:10 dilution of precipitated fibrils was used for the transfection. 5 μl of PTA-purified fibrils was diluted in 45 μl Opti-MEM to a final volume of 50 μl. The previous Opti-MEM mixture was added to 47 μl of Opti-MEM and 3 μl of Lipofectamine-2000 and incubated in room temperature for 2 hr as described in Safar et al., 1998 (link); Woerman et al., 2016 (link). The mixture was added to cells, washed after 18 hr and re-plated before analysis exactly as mentioned previously.
For PTA-precipitated brain homogenate, 1:10 dilution of precipitated fibrils was used for the transfection. 5 μl of PTA-purified fibrils was diluted in 45 μl Opti-MEM to a final volume of 50 μl. The previous Opti-MEM mixture was added to 47 μl of Opti-MEM and 3 μl of Lipofectamine-2000 and incubated in room temperature for 2 hr as described in Safar et al., 1998 (link); Woerman et al., 2016 (link). The mixture was added to cells, washed after 18 hr and re-plated before analysis exactly as mentioned previously.
6
Imaging of CASK Aggregation in HEK-293 Cells
7
Neurexin1-β and CASK Colocalization
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