Human neuroblastoma SH-SY5Y cells were maintained in DMEM/F-12 supplemented with 10% low-endotoxin fetal bovine serum, 2 mM L-glutamine, and 1% penicillin-streptomycin (all from Gibco, Thermo Fisher Scientific, USA). For experiments, cells were plated on clear bottom 96-well plates (Phenoplate, Perkin Elmer, USA) at 6 × 104 cells/well and maintained as described in a previous study (Westbroek et al., 2016 (link)) for 24 h to allow the cells to attach. WT and GBA1 knockout (KO) immortalized neural cells were kindly provided by Ellen Sidransky from the National Institutes of Health, and the generation and characterization of these cells have been described previously (Westbroek et al., 2016 (link)). These cells were cultured in neurobasal growth media supplemented with 2% B27 supplement, 1 mM L-glutamine, and 100 U/mL penicillin-streptomycin (all from Thermo Fisher Scientific, USA) in T75 flasks (Thermo Fisher Scientific, USA) pre-coated with poly-L-lysine (Sigma-Aldrich, USA) at 37°C with 5% CO2. For live cell imaging experiments, 2.5 × 104 cells per well were plated on clear bottom 96-well plates (Phenoplate, PerkinElmer, USA) pre-coated with poly-L-lysine and incubated at 37°C with 5% CO2 until confluent.
Phenoplate
Manufactured by PerkinElmer
Sourced in United States
The PhenoPlate is a lab equipment product by PerkinElmer. It is a high-throughput system designed for automated cell-based assays and screening applications.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Operetta cls, by PerkinElmer (2 mentions)
Cyclopamine, by Cayman Chemical (2 mentions)
Sb431542, by Cayman Chemical (2 mentions)
Xav939, by Cayman Chemical (2 mentions)
Y 27632, by Bio-Techne (2 mentions)
Ensight multimode plate reader, by PerkinElmer (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Nucblue, by Thermo Fisher Scientific (1 mentions)
Harmony software, by PerkinElmer (1 mentions)
L7 hpsc passaging solution, by Lonza (1 mentions)
N2 and b27 supplements, by Thermo Fisher Scientific (1 mentions)
Stempro ezpassage tool, by Thermo Fisher Scientific (1 mentions)
Opera phenix high content confocal imaging system, by PerkinElmer (1 mentions)
Ibl 437c, by PerkinElmer (1 mentions)
Operetta high content screening system, by PerkinElmer (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Low endotoxin fetal bovine serum, by Thermo Fisher Scientific (1 mentions)
T75 flask, by Thermo Fisher Scientific (1 mentions)
10 protocols using phenoplate
1
SH-SY5Y Cell Culture and Preparation
2
Cytotoxicity Assay in CFBE41o- Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CFBE41o- cells were seeded in 96-well plates (PhenoPlate, PerkinElmer) at a density of 20,000 cells per well. After 24 h, cells were treated for further 24 h with PP028, VX-445, and doxorubicin at various concentrations. Finally, cells were labeled with NucBlue (Invitrogen, Live Cell Stain ReadyProbes reagent). The images were acquired with the High Content Analysis System, OperettaCLS (PerkinElmer) and analyzed by Harmony Software (PerkinElmer).
3
Directed Differentiation of iPSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
iPSC lines were passaged using Accutase (Innovative cell) and replated onto Geltrex-coated (1:50 dilution in DMEM/F12) 12-well plates at 7–8 × 105 cells/well in mTeSR1 with 10 μM rho-kinase inhibitor (Y-27632; Tocris, 1254). The following day, or when the cells reached 80–100% confluency, the medium was then changed to 3N (50:50 DMEM/F12:neurobasal with N2 and B27 supplements) without vitamin A with 2 μM DMH1 (Tocris, 4126), 2 μM XAV939 (Cayman Chemical, 13596), and 10 μM SB431542 (Cayman Chemical, 13031) (2 mL of medium per well). 75% of this medium was changed daily with 1 μM cyclopamine (Cayman Chemical, 11321) added beginning on day 1. On day 4, the monolayer was cut into squares using the StemPro EZ passage tool (Gibco). The squares were incubated for 2 minutes with L7 hPSC passaging solution (Lonza). After aspirating the L7 solution, the squares were sprayed off the bottom with 1 mL of preconditioned culture media with a P1000 micropipette. An additional 1 mL of fresh culture medium with the 4 inhibitors was added. Approximately 100 μL of resuspended monolayer squares were then transferred into the wells of black-walled thin-bottom 96-well plates (Greiner μClear or PerkinElmer PhenoPlate) preincubated at 37 °C for 30 minutes with 35 μL of 100% Geltrex solution in each well.
4
Cellular Uptake of Lipoproteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HepG2 cells were seeded (40,000 cells/well) on 96-well plates (PhenoPlate, PerkinElmer), serum-deprived (0.5% FBS) for 16 hr and then treated with α-MSH or LD211 for 18 hr at 37 °C. After the treatment, cells were washed with PBS and incubated with fluorescently-labeled HDL (Dil-HDL, 20 µg/ml, CliniSciences) or LDL (Dil-LDL, 10 µg/ml, CliniSciences) for 4 hr at 37 °C. After the incubation, cells were again washed with PBS and the fluorescence signal was measured with EnSight Multimode Plate Reader (PerkinElmer) with the 549 nm excitation and 565 emission wavelengths and normalized against cell confluency.
5
Quantifying Apoptosis in Cultured gADSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The percentage of cultured apoptotic gADSCs in four different BCMs was assessed utilising Annexin V/PI staining. gADSCs were seeded at a density of 1 × 104 cells/well in a 96-well, black-walled, optically clear flat-bottom tissue-culture treated dish (PhenoPlate, PerkinElmer). After 48 h, the culture medium was aspirated, and cells were washed with PBS. Subsequently, cells were stained using the Dead Cell Apoptosis Kit with Annexin V Alexa Fluor 488 and PI (Thermofisher Scientific) according to the manufacturer's guidelines. In brief, cells were rinsed with ice-cold PBS and then incubated in 200 μl of binding buffer. Ten microliters of Annexin V stock solution were added to the cells and incubated for 15 min at room temperature. The cells were then further incubated with 2 μl propidium iodide (PI) and counterstained with Hoechst 33342 for 10 min for nuclear visualisation. The high-content analysis system Operetta CLS (PerkinElmer) captured and analysed the images using Harmony 5.1 software (PerkinElmer).
6
iPSC Differentiation to Neural Progenitors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We Accutase passaged 7–8 × 105 iPSC lines onto Geltrex-coated (1:50 dilution in DMEM/F12) 12-well plates in mTeSR1 with 10 µM Y-27632 (Tocris, Bristol, UK). When the cells reached 80–100% confluency, the medium was changed to 3N (50:50 DMEM/F12:neurobasal with N2 and B27 supplements [Thermo]) without vitamin A with 2 µM DMH-1 (Tocris), 2 µM XAV939 (Cayman Chemical, Ann Arbor, MI, USA), and 10 µM SB431542 (Cayman Chemical) with 2 mL of medium per well. Subsequently, 1.5 mL medium were changed daily with 1 µM cyclopamine (Cayman Chemical) added beginning on day 1. On day 4, the monolayer was cut into squares using the StemPro EZ passage tool (Thermo). The squares were incubated for 2 min with the hypertonic sodium citrate solution. After aspiration, the squares were sprayed off the bottom with 1 mL of preconditioned culture media with a P1000 micropipette. An additional 1 mL of fresh culture medium with the 4 inhibitors was added. Approximately 100 µL of resuspended monolayer squares were then transferred into the wells of black-walled thin-bottom 96-well plates (µClear [Greiner Bio-One, Monroe, NC, USA] or Phenoplate [PerkinElmer, Waltham, MA, USA]) preincubated at 37 °C for 30 min with 35 µL of 100% Geltrex solution in each well.
7
High-Throughput Multicolor Cell Screening
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For screening of the multicolour cell pool of 41 HAP1 clones, cells were seeded in 12 96-well imaging plates (PerkinElmer PhenoPlate) at a concentration of 2,500 cells per well. At 56 h after seeding, 100 FOVs (approximately one-third of the entire well area; each FOV has a resolution of 1,080 × 1,080 pixels) were imaged per well with an Opera Phenix high-content confocal imaging system (PerkinElmer) using a ×40 water immersion objective. For the treatment of cells with compounds, medium was added to compound plates for pre-diluting compound stocks, before transferring pre-diluted compounds to imaging plates for a final compound concentration of 10 µM and 0.1% DMSO for the majority of compounds. At 6 h after treatment, the same FOVs in compound-treated wells were imaged again as described above.
8
Quantifying DNA Damage Foci by Microscopy
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
15.000 cells were seeded in Phenoplate (6,055,302, PerkinElmer) black well plates and the following day cells were gamma-irradiated at 5 Gy with an IBL-437C (CIS Biointernational). Control plate was left untreated. Cells were fixed with 4% PFA. Afterwards, blocking and permeabilization was performed with PBS/5% BSA/0,3% Triton-X during 1 h at RT. Staining with the primary antibody p-γH2AX (9718 T, CST) dissolved in PBS/1% BSA/0,3% Triton-X was performed overnight at 4ºC. The following day, cells were washed × 3 with PBS and secondary anti-rabbit Alexa Fluor 647 (A21244, Invitrogen) was added during 2 h at RT in the dark. Cells were washed again × 2 with PBS and incubated with 1 μg/ml DAPI (D9542, Sigma) for 10 min. After washing, cells were imaged with the Opera or Operetta High Content Screening System (Perkin Elmer), using the 40 × objective. Segmentation of the nuclei using the DAPI signal and quantification of the number of p-γH2AX foci per cell was done using Harmony® High-Content Imaging and Analysis Software.
9
Imaging Co-cultured Ovarian Cancer Cells and Cancer-Associated Fibroblasts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
4.5x103 immortalised GFP expressing omCAFs or 4.5x103 KURAMOCHI or COV318 or 0.9x103 OVCAR4 or OVCAR8 HGS ovarian cancer cells labelled with 1 µM CMPTX cell tracker red (Invitrogen) (both omCAF and cancer cell nuclei were labelled with 20 µM Hoechst) were plated per well of a 96 well PhenoPlate (Perkin Elmer) in either RPMI (KURAMOCHI, OVCAR4, OVCAR8) or DMEM (COV318) containing 30 µg/ml OmGel (mix of OmGels derived from 6 HGS ovarian cancer patients) or Matrigel (growth factor reduced, BD Biosciences) supplemented with 4 % FBS, 1 % penicillin/streptomycin and 1 % glutamine (all from Life Technologies). Cells were plated on plastic wells not coated or pre-coated with either 30 µg/ml OmGel or growth factor reduced Matrigel. Images were taken on an Opera Phenix high-throughput confocal microscope (Perkin Elmer) at 37 °C and 5 % CO2. Nine images were taken per well using 10x objective with sequential excitation at 405 nm, 488 nm, 561 nm. Images were taken every 24 hrs over a 96 hr period. To quantify cell number, analysis was done using Harmony 4.9 Software (Perkin Elmer) to segment nuclei and cells. Supervised machine learning using Harmony’s “Phenologic” function, allowed cells to be classified into three observed, morphological categories.
10
Quantifying Cholesterol Levels in HepG2 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HepG2 cells were seeded (40,000 cells/well) on 96-well plates (PhenoPlate, PerkinElmer) and grown until the cells reached 70% confluency. Thereafter, cells were first serum-deprived (0.5% FBS) for 16 hr, and then treated with either α-MSH or LD211 for 24 hr. After the treatment, cells were washed with PBS and fixed with 4% paraformaldehyde (Sigma-Aldrich) for 15 min at room temperature. Cells were subsequently washed with PBS and incubated with 1.5 mg/ml glycine (Sigma-Aldrich, 10 min at RT) to quench unreacted paraformaldehyde followed by washing with PBS. Cells were stained with 50 ug/ml of Filipin (Sigma-Aldrich, #F9765) for 1 hr at 37 °C and washed with PBS. Fluorescence signal was measured with EnSight Multimode Plate Reader (PerkinElmer) with the 360 nm excitation and 480 emission wavelengths.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!