The hiPSC line used has been generated and fully characterized in Luni et al. (2016) (link). hiPSCs were maintained as feeder-free cells in TeSR-E8 (TeSR-E8 Basal Medium – STEMCELL Technologies, #05941) medium in 6-well plates coated with 0.5% of Matrigel (Corning Matrigel Matrix – Growth Factor Reduced, SACCO – Corning, #354230). Upon reaching 70% confluence, cells were dissociated in 0.5 nM EDTA and replated at 1:6–1:10 ratio. For neural cells differentiation, hiPSCs were dissociated at single cell level with TrypLE enzyme (TrypLE Select Enzyme (1X), no phenol red – Thermo Fisher Scientific, # 12563011) and seeded in the microfluidic chambers coated with 1% Matrigel.
Tryple select enzyme 1x
Manufactured by Thermo Fisher Scientific
Sourced in Australia
TrypLE™ Select Enzyme (1X) is a recombinant trypsin-like protease designed for gentle cell detachment and dissociation. It is a ready-to-use, liquid enzymatic cell dissociation reagent.
Automatically generated - may contain errors
Lab products found in correlation
Panc 1, by American Type Culture Collection (2 mentions)
Aspc 1, by American Type Culture Collection (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Tesr e8 basal medium, by STEMCELL (1 mentions)
Bxpc 3, by American Type Culture Collection (1 mentions)
Capan 1, by American Type Culture Collection (1 mentions)
Cellartis def cs culture system, by Takara Bio (1 mentions)
Mtesr1 plus medium, by STEMCELL (1 mentions)
Hygromycin b, by Thermo Fisher Scientific (1 mentions)
Y 27632, by Selleck Chemicals (1 mentions)
Fugene hd transfection reagent, by Promega (1 mentions)
Foetal bovine serum (fbs), by Merck Group (1 mentions)
96 well ultra low attachment plate, by Thermo Fisher Scientific (1 mentions)
Sh peg2k nh2, by Merck Group (1 mentions)
Amphotericin b, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium d5796, by Merck Group (1 mentions)
Hoechst 33258, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Trisodium citrate, by Merck Group (1 mentions)
Gold 3 chloride trihydrate, by Merck Group (1 mentions)
12 protocols using tryple select enzyme 1x
1
Feeder-Free Culture and Neural Differentiation of hiPSCs
2
Pancreatic Cancer Cell Line Cultivation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PANC-1, CAPAN-1, BxPC-3, and AsPC-1 pancreatic cancer cell lines were purchased from American Type Culture Collection (ATCC) (Manassas, USA). Cell lines were selected based on their variable MUC1-CE expression, ranging from strong (PANC-1), moderate (CAPAN-1), moderate-weak (BxPC-3) and weak (AsPC-1) levels (Hull et al. 2020 (link)). PANC-1 cells were cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% foetal calf serum (FCS) and 1% penicillin/streptomycin (P/S). CAPAN-1 cells were cultured in Iscove’s Modified Dulbecco’s Medium supplemented with 20% FCS and 1% P/S. BxPC-3 and AsPC-1 cells were cultured using Roswell Park Memorial Institute 1640 medium supplemented with 10% FCS and 1% P/S. For all experiments, cells were grown to confluence in T75 flasks in a 5% carbon dioxide in air environment set to 37 °C. At confluence, cells were washed twice with DPBS and detached from the flasks using TrypLE™ Select Enzyme (1X) (Thermo Fisher Scientific Australia Pty Ltd, Scoresby, Australia). All cell lines were used within three months of resuscitation.
3
iPSC Generation from Cord Blood
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human iPSC cell OARSAi002-A was previously generated in our laboratory from the cord blood of a healthy non-Hispanic white male using self-replicative RNA reprogramming technology (15 (link)). Cells were maintained as a single-cell culture (no colony formation) on COAT-1 pre-coated 6-well tissue culture plates in the Cellartis DEF-CS Culture System (Takara Bio, Inc.) at 37˚C in a humidified incubator supplied with 5% CO2. For passaging, cells were dissociated into single cells with TrypLE Select Enzyme (1X) (Thermo Fisher Scientific, Inc.) and seeded at a density of 4-5x104 cells/cm2. The cell culture medium was changed daily until the cells reached a confluence of ~1.5-3.0x105 cells/cm2, which generally occurred 3-4 days post passage. The pluripotency of the iPSCs was routinely assessed using immunocytochemistry staining of major pluripotency protein markers, including octamer-binding transcription factor 4 (OCT4), SRY (sex determining region Y)-box (SOX)2, stage-specific embryonic antigen-4 (SSEA4) and T cell receptor α locus (TRA-1-60) using a Pluripotent Stem Cell 4-Marker Immunocytochemistry Kit (cat. no. A24881) according to the manufacturer's protocol (Thermo Fisher Scientific, Inc.).
4
Generation of hESCs with Stable ZZZ3 Knockdown
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
hESCs with stable KD of ZZZ3 gene were generated by transfecting cells with PB transposon plasmids system with PB transposase expression vector pBase. Three PB U6-promoter-driven shRNAs against ZZZ3 mRNA or scramble control were purchased from Vector Builder. For DNA transfection, hESCs were dissociated as single cells using TrypLE Select Enzyme (1X) (Thermo Fisher Scientific), and 250,000 cells were co-transfected with PB constructs (550 ng) and pBase plasmid (550 ng) using FuGENE HD transfection reagent (3.9 μL) (Promega), following the manufacturer’s instruction. Cells were cultured onto Matrigel-coated 12-well plate in mTeSR1 Plus medium (STEMCELL Technologies, Vancouver, Canada) with 10 μM Y27632 (ROCKi, Rho-associated kinase inhibitor, Selleckchem). After 48 h, transfected cells were selected with hygromycin B (200 μg/mL, Thermo Fisher Scientific) diluted in mTeSR1 Plus for 2 weeks before performing experiments. The sequences of PB ZZZ3 shRNA are provided in the Table S3 .
5
Gold Nanoparticles Synthesis and Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Gold (III) chloride trihydrate (HAuCl4·3H2O, ≥99.9% trace metals basis), tri-sodium citrate (for molecular biology, ≥99%), tannic acid (ACS reagent), SH-PEG2k-NH2, Foetal Bovine Serum (FBS), Amphotericin-β, Penicillin-Streptomycin, and Dulbecco’s Modified Eagle’s Medium (D5796) were all acquired from Sigma-Aldrich, Johannesburg, South Africa. TrypLE™ Select Enzyme (1X) (12563-029) ThermoFisher, Johannesburg, South Africa). Hoechst 33258, Caspase 3, and 9 Multiplex Assay kit (Ab219915) as well as 96-well ultra-low attachment plates (174929) were purchased from ThermoFisher, Johannesburg, South Africa. Annexin V/PI apoptosis detection kit (556570) was procured from BD Biosciences, The Scientific Group, Johannesburg, South Africa).
6
Culturing Human Pancreatic Cancer Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The human pancreatic cancer cell lines PANC-1 and AsPC-1 were purchased from American Type Culture Collection (Manassas, VA, USA). PANC-1 cells were cultured using Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% foetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). AsPC-1 cells were cultured using Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% FBS and 1% P/S. All cell lines were grown in T75 flasks and incubated at 37 °C with 5% carbon dioxide in atmospheric oxygen. Cells were used within three months of resuscitation and regularly monitored for mycoplasma contamination using MycoAlert detection kit (Lonza Group Ltd., Basel, Switzerland). Unless otherwise stated, all cell culture reagents were purchased from Sigma-Aldrich Pty Ltd. (Castle Hill, Australia).
For experiments, cells were grown to confluence then washed twice with PBS and detached from the flask by TrypLE™ Select Enzyme (1X) (Thermo Fisher Scientific Australia Pty Ltd., Scoresby, Australia). Detached cells were centrifuged at 300× g for 5 min to separate the cell pellet and supernatant to prepare cells for seeding.
For experiments, cells were grown to confluence then washed twice with PBS and detached from the flask by TrypLE™ Select Enzyme (1X) (Thermo Fisher Scientific Australia Pty Ltd., Scoresby, Australia). Detached cells were centrifuged at 300× g for 5 min to separate the cell pellet and supernatant to prepare cells for seeding.
7
Quantitative Proteomic Analysis of Islet and Stem Cell Cultures
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
S6, S7d7, S7d14 and Wnt-modulated cell cultures were washed in DPBS and harvested with TrypLE™ Select Enzyme (1X) (12563011, Thermo Fisher Scientific), followed by centrifugation. Islets and hiPSC-derived cells were lysed as described previously (18 (link)). The protein concentration was determined using a BCA protein assay kit. Dry aliquots containing an estimated amount of 100 μg of proteins were further processed using Filter-Aided Sample Preparation (20 (link)). The six islet samples were combined and mixed to make a homogenous mixture, and 50 μg protein of the mix were aliqvoted into six separate samples for downstream TMT11-plex analysis.
8
Wnt5A-Induced Proteomics in Differentiated Cells
9
Characterization of hiPSCs and hiPSC-CMs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HiPSCs and hiPSC-CM were dissociated as single cells using TrypLE™ Select Enzyme (1x) (Gibco) and TrypLE™ Select Enzyme (10x) (Gibco), respectively. For hiPSCs 2 × 105 cells were fluorescently labelled for pluripotent stem cell surface markers using 1:20 anti-SSEA4-VioBlue (Miltenyi Biotec, Bergisch Gladbach, Germany) 130-098-366) and 1:600 anti-Tra1-60-Vio488 (Miltenyi Biotec, 130-106-872) antibodies. Then, cells were fixed and permeabilized using the FoxP3 Staining Buffer Set (Miltenyi Biotec, 130-093-142) and labelled for nuclear markers using 1:50 anti-Oct3/4-APC (Miltenyi Biotec, 130-123-318) and 1:100 anti-Nanog-PE (Cell Signaling, Danvers, MA, USA, 14955S). For the characterization of cardiac markers 2 × 105, hiPSC-CMs were fixed and permeabilized similarly to the hiPSCs and fluorescently labelled using 1:50 anti-cardiac Troponin T-FITC (Miltenyi Biotec, 130-119-575) and 1:10 anti-MLC2v-APC (Miltenyi Biotec, 130-106-134) antibodies. Isotype stainings were performed as controls. Cells were measured for marker expression using a MACSQuant VYB (Miltenyi Biotec) flow cytometer and data analyzed using FlowJo software.
10
Organoid Treatment and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Organoids treated with 0.15 mg/mL FDP-DOX and FDP-NV were harvested at 2, 6, 24 and 48 h. Briefly, media containing the particles were removed and the wells washed twice with 1X DPBS (Sigma Inc.) to remove any residual FDP particles. Organoids were harvested and dissociated into single cells by enzyme digest with TrypLE™ Select Enzyme (1X) (GIBCO) for 10 min at 37°C, followed by gentle mechanical pipetting and trituration with a 27 G 1¼ inch needle. Cells’ pellets were resuspended in 1X PBS supplemented with 2% FBS, and the suspension cell culture was collected into a 35 μm strainer tube and stained with DAPI (4ʹ,6-diamidino-2-phenylindole, ThermoFisher, Sci.) for 10 min. Cells were sorted with the LSRFortsea X-20 and the results analyzed with the FlowJo v10.7 software.
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!