hiPSCs were maintained in mTeSR™ Plus Basal Medium (Cat. No.100–11300, STEMCELL Technologies) + Primocin (Cat. No.NC9392943, Invivogen) in an incubator at 37°C with 5% CO2.. Cells were cultured on plates incubated with 10 μg/mL Vitronectin XF (Cat. No.07180, STEMCELL Technologies) for at least 1 hour at room temperature prior to passaging. Media was changed every other day, and cells were passaged mechanically or as single cells (10,000 cells/cm2) every 4–7 days. For generating single cell suspensions of hiPSCs, cells were dissociated with Accutase (Cat. No.# 07920_C, STEMCELL Technologies) for 3–5 minutes in a 37°C incubator. Accutase was diluted 1:1 with mTeSR Plus, 100ug/ml Primocin, and 10 μM Y-27632 (Cat. No.SM-0013-0010, Biological Industries, USA) and cells were centrifuged at 210 g for 5 minutes. For TIRF imaging, cells were seeded at 13,000 cell/cm2 density on Vitronectin XF coated MatTek dishes (Cat. No.P35G-1.5-14-C, MatTek Corporation Cells were regularly checked for mycoplasma contamination and karyotypic abnormalities.
Mtesr plus basal medium
Manufactured by STEMCELL
Sourced in France
MTeSR Plus Basal Medium is a culture medium designed for the maintenance and expansion of human pluripotent stem cells. It provides the necessary components for supporting the growth and self-renewal of these cells in vitro.
Automatically generated - may contain errors
Lab products found in correlation
B27 supplement, by Thermo Fisher Scientific (2 mentions)
Primocin, by STEMCELL (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Rna clean concentrator 5 kit, by Zymo Research (1 mentions)
Mtesr plus supplement, by STEMCELL (1 mentions)
Cytotune ips 2.0 sendai reprogramming kit, by Thermo Fisher Scientific (1 mentions)
Relesr reagent, by STEMCELL (1 mentions)
Hela cell, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Hela cells, by Roche (1 mentions)
D35c4 20 1.5 n, by Cellvis (1 mentions)
Mtesr plus 5x supplement, by STEMCELL (1 mentions)
X tremegene hp, by Roche (1 mentions)
Mem non essential amino acid, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Lonza (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
6 protocols using mtesr plus basal medium
1
Maintenance and Passaging of hiPSCs
2
Generating ATRT-like Model from Epi-iPSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Dual knockout Epi-iPSCs were subjected to neural induction to obtain an ATRT-like model. Before the induction, cells were maintained in mTeSR Plus Basal Medium (STEMCELL, Cat#: 100–0274) with antibiotics and 1x mTeSR Plus Supplement (STEMCELL, Cat#: 100–0275). On day 0, cells were dissociated by Accutase®, and 5 × 104 cells were seeded per well of a low attachment 24-well plate (Corning, Cat#: 3473) in DMEM/F-12 (ThermoFisher, Cat#: 11320033) containing 2% B-27 supplement (ThermoFisher, Cat#: 17504044), 100 U/mL penicillin, 100 μg/mL streptomycin, 10 μM transforming growth factor (TGF)-beta inhibitor, SB431542, and 10 μM ROCKi, Y-27632. After 48 h, the media was changed, and 50 ng/mL FGF2 was added to promote neural induction for spheroid formation until day 7 for characterization.
For RT-PCR, day-7 spheroids were collected and centrifuged at 300×g for 5 min. Then, the total mRNA was isolated from the spheroids using RNeasy® Mini Kit (Qiagen, Valencia, CA), concentrated, and cleaned using the RNA Clean & Concentrator-5 kit (Zymo, Irvine, CA).
For immunocytochemistry, the spheroids were collected and treated with Accumax® at 37 °C for 15 min. The dissociated cells were replated on a Matrigel-coated surface for 48 h before being fixed and probed for markers of interest.
For RT-PCR, day-7 spheroids were collected and centrifuged at 300×g for 5 min. Then, the total mRNA was isolated from the spheroids using RNeasy® Mini Kit (Qiagen, Valencia, CA), concentrated, and cleaned using the RNA Clean & Concentrator-5 kit (Zymo, Irvine, CA).
For immunocytochemistry, the spheroids were collected and treated with Accumax® at 37 °C for 15 min. The dissociated cells were replated on a Matrigel-coated surface for 48 h before being fixed and probed for markers of interest.
3
Reprogramming UDCs to iPSCs Using Sendai Kit
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
UDCs at passage below 5 were reprogrammed when cells reached a 30-60% confluency. To reduce the risk of genetic abnormalities, UDCs were reprogrammed to iPSCs using the non-integrative CytoTune™-iPS 2.0 Sendai Reprogramming Kit (Thermo Fisher, Waltham, MA, USA) (Figure 2A
4
Parkinson's Disease iPSC Neuronal Culture
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HeLa cells [American Type Culture Collection (ATCC)] were cultured in Dulbecco’s modified Eagle’s medium (Gibco; 11995-065) supplemented with 10% (v/v) heat-inactivated fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 μg/ml). Human iPSCs derived from a PD patient harboring a parkin loss-of-function mutation (B125: homozygous c.1072del, male, age of onset: 43) as well as a CRISPR-generated isogenic control were maintained on Cultrex-coated (R&D Systems; 3434-010-02) six-well plates, cultured in mTeSR Plus Basal Medium (STEMCELL Technologies; 100-0274) with mTeSR Plus 5X Supplement (STEMCELL Technologies; 100-0275) and were passaged every 5 to 6 days. All cells were maintained at 37°C in a 5% CO2 incubator and were previously verified by cytochrome c oxidase subunit I and short tandem repeat testing and tested for mycoplasma contamination. HeLa cells were transfected using X-tremeGENE HP (Roche; 6366546001). iPSC-derived dopaminergic neurons were transduced with the lentiviral constructs described below. For live-cell imaging, cells were grown on four-chamber glass bottom dishes (Cellvis; D35C4-20-1.5-N).
5
Comprehensive Cell Culture Media Formulations
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Leishmania culture medium: Equal parts SM and SDM-79 medium [30 (link),31 (link)] (pH 7.4) supplemented with 10% heat-inactivated FCS and 2 μg/mL hemin. mTeSR Plus medium: mTeSR Plus Basal Medium (pH 7.4) (STEMCELL Technologies) supplemented with mTeSR Plus 5x Supplement and 1% penicillin/streptomycin (Gibco). Complete RPMI medium: RPMI 1640 Glutamax (pH 7.4) (Gibco) supplemented with 10% heat-inactivated FCS (Gibco), 1% sodium pyruvate (Gibco), 1% penicillin/streptomycin, 25 mM HEPES (Gibco), 0.055 mM β-mercaptoethanol (Gibco). MDM medium: DMEM (pH 7.4) (Gibco) supplemented with 10% FBS, 1% Glutamax (Gibco), 0.055 mM β-mercaptoethanol, 1x MEM Non-Essential Amino Acids (Gibco) and 1% penicillin/streptomycin. X-Vivo 15 medium: X-Vivo 15 (pH 7.4) (Lonza) supplemented with 1% Glutamax, 0.055 mM β-mercaptoethanol and 1% penicillin/streptomycin.
6
Generation and Characterization of Episomal iPSCs and ATRT Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human episomal iPSC (Epi-iPSC, ThermoFisher, Cat#: A18945) expresses seven factors (SOKMNLT; SOX2, OCT4, KLF4, MYC, NANOG, LIN28, and SV40L T antigen), has no footprints, and is free of all reprogramming genes. The cells were cultured using mTeSR™ Plus Basal Medium (STEMCELL Technologies, Cat#: 100–0274) and passaged when they reached 80% confluency using Accutase® (Innovative Cell Technologies, Cat#: 10210-214). The cells were split into a 1:8–1:10 ratio for new culture per well of a 6-well plate with the rho-associated, coiled-coil containing protein kinase inhibitor (ROCKi) Y-27632 (10 μM, Sigma) for 24h.
The patient-derived ATRT cell lines used were CHLA-02-ATRT (ATCC, Cat#: CRL-3020) from a 20-month-old male and CHLA-05-ATRT (ATCC, Cat#: CRL-3037) from a 2-year-old male. Both cell lines were cultured in ultra-low attachment plates. The base medium for both the cell lines was Dulbecco's Modified Eagle Medium (DMEM)/Nutrient Mixture F-12 (DMEM/F12) (ATCC, Cat#: 30–2006) with 1x B-27 Supplement (Gibco, Cat#: 17504044), 20 ng/mL epidermal growth factor (EGF) (Gibco, Cat#: PHG0311), and 20 ng/mL fibroblast growth factor 2 (FGF2) (Gibco, Cat#: PHG0021). Accumax® (Innovative Cell Technologies, Cat#: AM-105) was used to dissociate aggregates of these ATRT cell lines.
The patient-derived ATRT cell lines used were CHLA-02-ATRT (ATCC, Cat#: CRL-3020) from a 20-month-old male and CHLA-05-ATRT (ATCC, Cat#: CRL-3037) from a 2-year-old male. Both cell lines were cultured in ultra-low attachment plates. The base medium for both the cell lines was Dulbecco's Modified Eagle Medium (DMEM)/Nutrient Mixture F-12 (DMEM/F12) (ATCC, Cat#: 30–2006) with 1x B-27 Supplement (Gibco, Cat#: 17504044), 20 ng/mL epidermal growth factor (EGF) (Gibco, Cat#: PHG0311), and 20 ng/mL fibroblast growth factor 2 (FGF2) (Gibco, Cat#: PHG0021). Accumax® (Innovative Cell Technologies, Cat#: AM-105) was used to dissociate aggregates of these ATRT cell lines.
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!