Islets were cultured with EdU at a concentration of 10 μM, with daily media changes, for 96 hours [11 (link)]. Pools of 50 islets were dispersed and plated on poly-D lysine coated coverslips (BD). EdU was detected using AlexaFluor 555 EdU cell proliferation kit (Invitrogen). Cells were counterstained with insulin to define β-cells and DAPI to define nuclei. Five sections containing greater than 400 nuclei were evaluated from EdU signal evaluated using ImageJ software for each experimental condition.
Poly d lysine coated coverslip
Manufactured by BD
Sourced in United States
Poly-D-lysine coated coverslips are a type of laboratory equipment used for cell culture applications. They provide a positively charged surface that promotes cell attachment and growth. The coating is made of the synthetic amino acid polymer, poly-D-lysine, which enhances the adherence of cells to the glass coverslip substrate.
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Lab products found in correlation
Human plasmin, by Merck Group (3 mentions)
Ames bicarbonate solution, by Merck Group (3 mentions)
Eclipse microscope, by Nikon (2 mentions)
H4a3 sc 20011, by Santa Cruz Biotechnology (2 mentions)
Alexa fluor 488 goat anti rabbit, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 594 goat anti mouse, by Thermo Fisher Scientific (2 mentions)
Vectashield mounting medium, by Vector Laboratories (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Papain, by Roche (2 mentions)
Uridine, by Merck Group (2 mentions)
Collagenase d, by Roche (2 mentions)
Collagenase a, by Roche (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Nb b27, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
B27 supplement, by Thermo Fisher Scientific (1 mentions)
Tamoxifen, by Merck Group (1 mentions)
Prism 7, by GraphPad (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Cell Signaling Technology (1 mentions)
21 protocols using poly d lysine coated coverslip
1
Measuring β-cell Proliferation with EdU
2
Evaluating in vivo efficacy of BMP inhibitors
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For examining in vivo efficacies, stock solutions of LDN193 (in DPBS, 100 mg/ml) and LDN214 (in DMSO, 100 mg/ml) were diluted using MCT and administrated to mice by oral gavage. Tamoxifen (T5648; Sigma-Aldrich) was dissolved at a concentration of 20 mg/ml in corn oil and administrated by oral gavage at 200 mg/kg. For survival analyses, mice after treatment with Tamoxifen or BMP inhibitors were monitored daily and sacrificed when they exhibited brain tumor symptoms (ataxia and tilted heads). Log-rank survival analyses were performed in GraphPad Prism 7.
To isolate tumor cells, tumor tissue from Ptch1-deficient mice were digested in a papain solution to obtain a single-cell suspension and then centrifuged through a 35 and 65% Percoll gradient. Cells from the 35–65% interface were suspended in DPBS plus 0.5% BSA. Cells were then suspended in NB-B27 (neurobasal with 1 mM sodium pyruvate, 2 mM L-glutamine, B27 supplement, and 1% penicillin/streptomycin, all from Invitrogen) and plated on poly-D-lysine–coated coverslips (BD Biosciences). Human recombinant BMP2, BMP5, and BMP7 (Peprotech) were used at 80 ng/ml for in vitro treatment of tumor cells.
To isolate tumor cells, tumor tissue from Ptch1-deficient mice were digested in a papain solution to obtain a single-cell suspension and then centrifuged through a 35 and 65% Percoll gradient. Cells from the 35–65% interface were suspended in DPBS plus 0.5% BSA. Cells were then suspended in NB-B27 (neurobasal with 1 mM sodium pyruvate, 2 mM L-glutamine, B27 supplement, and 1% penicillin/streptomycin, all from Invitrogen) and plated on poly-D-lysine–coated coverslips (BD Biosciences). Human recombinant BMP2, BMP5, and BMP7 (Peprotech) were used at 80 ng/ml for in vitro treatment of tumor cells.
3
Immunofluorescent Localization of mTOR and LAMP1 in Primary Macrophages
4
Macaque Retinal Tissue Preparation and Electrophysiology
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We obtained retinal tissue from macaque monkeys (Macaca nemestrina, Macaca mulatta, or Macaca fascicularis) via the tissue distribution program at the Washington National Primate Research Center. All procedures were approved by the Institutional Animal Care and Use Committee at the University of Washington. Results did not depend obviously on age (2 to 18 y) or sex, although our ability to discern such differences is quite limited. Dissection procedures have been described previously (8 (link)). After enucleation, the eye was hemisected and the vitreous humor was removed mechanically, sometimes assisted by treatment with human plasmin (∼50 g/mL; Sigma-Aldrich or Haematologic Technologies Inc.). The retina was dark adapted for ∼1 h, and all subsequent procedures were performed under infrared light using night-vision goggles. The retina and pigment epithelium were separated from the sclera and stored in oxygenated (95%O2/5%CO2) Ames bicarbonate solution (Sigma-Aldrich) in a light-tight container. Retinal mounts with the pigment epithelium attached were placed onto poly-D-lysine–coated coverslips (BD Biosciences) with the RGCs facing up. During experiments, retinal tissue was perfused at 7 to 9 mL/min with Ames solution at ∼32 °C. Recordings were made from the peripheral retina with an average eccentricity of 25° ± 10° (mean ± SD).
5
Immunofluorescent Localization of mTOR and LAMP1 in Primary Macrophages
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Primary human macrophages were cultured on poly-D lysine-coated coverslips (BD Biosciences Discovery Labware) for 24 h in the presence or absence of IFN-γ (100 U/ml). Cells were fixed with 2% formaldehyde for 15 min at room temperature and were permeabilized with 0.1% Tween-20 (PBST) at room temperature for 10min. 5% goat serum (Santa Cruz (sc-2043)) was used for blocking at 37 °C for 1 h, and then cells were stained with rabbit antibody to mTOR (Cell Signaling 2983 (7C10)), and mouse antibody to LAMP-1 (Santa Cruz H4A3 sc-20011) simultaneously in the 4°C cold room for at least 12 h. Alexa Fluor 488 goat anti-rabbit (Invitrogen) and Alexa Fluor 594 goat anti-mouse (Invitrogen) secondary antibodies were then used to detect mTOR and LAMP1 primary antibodies, respectively. Coverslips were mounted with Vectashield mounting medium (Vector Laboratories) and images were obtained using a Nikon Eclipse Microscope.
6
Visualizing THAP10 Nuclear Localization
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Analysis for nuclear localization of endogenous THAP10 was performed in transfected cells [NIH3T3 cells transfected with pEGFP or pTHAP10‐EGFP; cells were grown on poly‐D‐lysine‐coated cover slips (BD Biosciences, San Jose, CA, USA) 24 h after transfection] or Kasumi‐1, HL‐60 and t(8;21) AML blasts. Briefly, cells were fixed for 20 min with 4% cold paraformaldehyde and then permeabilized for 5 min with 0.05% Triton X‐100. Cells were incubated with 10 μl of Hoechst33342 (100 mg/l, Thermo Fisher Scientific) for 15 min for transfected cells. For Kasumi‐1, HL‐60 and t(8;21) AML blasts, permeabilized cells were then blocked with PBS‐BSA (PBS with 1% bovine serum albumin) and incubated with THAP10 primary antibodies (2 μg/ml, NBP1‐86226, Novus Biologicals, USA) overnight at 4°. Cells were then washed three times in PBA‐BSA and incubated in the dark for 1 h with Alexa Fluor 488‐conjugated secondary antibodies (1/500, #4412, Cell Signalling Technology, USA) diluted in PBA‐BSA. After three PBS washes, nuclei were counterstained with DAPI (0.2 μg/ml, #4083, Cell Signalling Technology, USA). Following extensive washing in PBS, samples were air‐dried and mounted in Rubber Cement (Union Rubber, Inc., Trenton, NJ, USA). Immunofluorescence images were captured using confocal microscopy (LSM 880, ZEISS, Germany). Green, GFP‐THAP10 or endogenous THAP10; blue, nucleus.
7
Isolation and Staining of Neoblasts
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Neoblasts in G2/M phase (X1), or G0/G1 phase (X2), and differentiated cells (Xins) were isolated by Fluorescence-Activated Cell Sorting based on DNA content (Hoechst fluorescence) as reported by (23 (link)), following the procedures described previously (13 (link)).
For staining of the isolated cells, cell suspensions isolated by FACS were collected in CMFB and centrifuged at ∼300 g for 5 min at 4°C. Cells were washed in CMF, spotted onto poly-d -lysine coated coverslips (BD Biosciences), allowed to settle for ∼30 min, and fixed in 4% PFA (in PBS) for 20 min at room temperature. For SYTO RNAselect staining, fixation instead was performed for 10 min at −20°C in ice-cold methanol. Controls and treatment were always spotted on the same cover slip, and went through all staining steps using the same solutions in the same well. IF and FISH labelings were carried out similarly to the whole-mount protocol, with wash steps and antibody incubations shortened to 10 min and 1 hour, respectively.
For staining of the isolated cells, cell suspensions isolated by FACS were collected in CMFB and centrifuged at ∼300 g for 5 min at 4°C. Cells were washed in CMF, spotted onto poly-
8
Phospho-Syk Activation in mDCs
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mDC were stimulated for indicated time period on poly-D-lysine coated coverslips (BD), washed with cold PBS and stained for phospho-Syk (Tyr 525/526) (Cell Signaling, 2710). Coverslips were then mounted on slides with DAPI fluoromount G (Southern Biotech) and analyzed by confocal microscopy (Leica SP5).
9
Characterization of Cav1.2 and Cav1.3 Splice Variants
10
Immunofluorescence Imaging of D5 Dopamine Receptor
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