AdMSC/PBMC were cocultured for 3 days as in proliferation assays. PBMC were collected and adherent cells detached (Triple Select; Gibco, USA). For AdMSC and T cell purification, we first collected the cells that were in suspension from the co-culture (PBMC), washed and collected them again (Suspension A). Then, we washed adherent AdMSC with DPBS (Gibco, Rockville, USA), detached adherent them with Tryple Select (Gibco, Rockville, USA) and collected them separately (Suspension B). Then, we used the PanT cell kit (Miltenyi Biotec GmBH Bergisch Gladbach, Germany,) for magnetic negative selection of T cells (which contains the following antibodies anti- CD14, CD16, CD19, CD36, CD56, CD123 and CD235a) using suspensions A and B, separately. Therefore, all non-T cells such as monocytes, NK, B cells erythrocytes, granulocytes and dendritic cells remain attached to the column and, as T cells and MSC have none of these molecules, they do not adhere to the column. Then, to obtain T cells from Suspension A, we used the CD4+ and CD8+ kits for magnetic positive selection (Miltenyi Biotec GmBH Bergisch Gladbach, Germany). Cell purity: 70–90% (FACS). Isolated cells were stored in RNAprotect® Cell Reagent (Qiagen, AMBION,USA) at −80 °C, until RNA extraction.
Triple select
Manufactured by Thermo Fisher Scientific
Sourced in United States
TripLE Select is a cell dissociation reagent manufactured by Thermo Fisher Scientific. It is designed to dissociate cells from cell culture surfaces in a gentle and efficient manner.
Automatically generated - may contain errors
Lab products found in correlation
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Pan t cell kit, by Miltenyi Biotec (1 mentions)
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (1 mentions)
Tryple select, by Thermo Fisher Scientific (1 mentions)
Rnaprotect cell reagent, by Qiagen (1 mentions)
Lsrii facs analyzer, by BD (1 mentions)
Purmorphamine, by Cayman Chemical (1 mentions)
Dorsomorphin, by Merck Group (1 mentions)
Sb431542, by Cayman Chemical (1 mentions)
Fgf8b, by Thermo Fisher Scientific (1 mentions)
Dbcamp, by Merck Group (1 mentions)
Tgf β3, by R&D Systems (1 mentions)
Basic fibroblast growth factor (bfgf), by Nacalai Tesque (1 mentions)
Sb431542, by Thermo Fisher Scientific (1 mentions)
Chir99021, by Thermo Fisher Scientific (1 mentions)
Chir99021, by Cayman Chemical (1 mentions)
Y 27632, by Cayman Chemical (1 mentions)
Hepes, by Merck Group (1 mentions)
Brain derived neurotrophic factor (bdnf), by Thermo Fisher Scientific (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
11 protocols using triple select
1
Isolation of T Cells and AdMSC
2
Phenotyping Human Thymic Aggregates
3
Differentiation of hiPSCs into Dopaminergic Neurons
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Neuronal differentiation of human iPSCs was induced as previously described (Arioka et al., 2018 (link)). Briefly, iPSCs were pretreated with 3 µM SB431542 (Cayman, Ann Arbor, MI, United States), 3 µM CHIR99021 (Cayman), and 3 µM dorsomorphin (Sigma, Burlington, MA, United States) for 1 week (day 0–7) and dissociated into single cells by incubation with TripLE Select (Gibco) for 5 min. The cells were cultured in neurosphere medium containing MHM [DMEM/F12, supplemented with 1× N2 (Gibco), 0.6% glucose (Sigma), 5 mM HEPES (Sigma), 100 units/mL penicillin and 100 μg/mL streptomycin, 1× B27 (Gibco), 20 ng/mL bFGF, 10 ng/mL hLIF (Nacalai, Kyoto, Japan), 10 µM Y-27632 (Cayman), 3 µM CHIR99021, 2 μM SB431542, 100 ng/mL FGF8b (Peprotech, Cranbury, NJ, United States), and 1 µM purmorphamine (Cayman) for 1 week (day 7–14)]. On day 14, neurospheres were passaged by dissociation into single cells in the same manner as primary neurosphere formation. For terminal differentiation into dopaminergic neurons, secondary neurospheres were dissociated 1 week after passage (day 21) and plated onto poly-L-ornithine/laminin/fibronectin-coated dishes at the density of 3 × 104 cells/cm2 in dopaminergic neuron medium [MHM supplemented with B27, 10 µM DAPT (Cayman), 20 ng/mL BDNF (Peprotech), 20 ng/mL GDNF (R&D), 0.2 mM ascorbic acid (Sigma), 1 ng/mL TGF-β3 (R&D), and 0.5 mM dbcAMP (Sigma)].
4
Intercellular Junctions Evaluation in Cell Aggregates
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To evaluate the tightness of intercellular junctions in T24 and T24 Ncadlow cell aggregates, we performed hanging drop assay (adapted from [50 (link)]). For this, cancer urothelial cells T24 and T24 Ncadlow were harvested with TripleSelect (Gibco, Life Technologies), counted using a haemocytometer, and adjusted to a concentration of 2 × 106 cells/mL. 30 µl drops of cell suspension were deposited on the bottom of the lid of the 100 mm cell culture dish and the corresponding culture dish was filled with 10 mL sterile PBS. The PBS-filled culture dish was then covered with the lid, which resulted in hanging drops of cell suspension from the lid. Cells were incubated at 37 °C in a 95% humidified atmosphere of 5% CO2 in the air until cell aggregates have formed at the bottom of the drop (1–2 days). Cell–cell adhesion was assessed by counting the cells released from the cell aggregates after forceful pipetting (10 times with a 200 μL Eppendorf pipette tip cut widely 5–6 mm from the tip). For each of the cell lines, we conducted at least four independent experiments in quadruplicates.
5
Isolation and Characterization of Human Lymphatic Endothelial Cells
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Human LNSCs were harvested from a 6-well dish using 1 ml TripLE™ Select (Gibco) for 10 min at 37°C. Subsequently, cells were washed in PBA buffer (PBS containing 0.01% NaN3 and 0.5% bovine serum albumin (BSA) (Sigma Aldrich), and stained for 1 h with rat IgG2a anti-human Podoplanin (clone NZ-1, AngioBio, Huissen, the Netherlands) on ice. Afterwards cells were washed again in PBS buffer, followed by a second incubation for 30 min on ice protected from light using the following directly labeled antibodies: Polyclonal goat anti-rat IgG AlexaFluor647 (Invitrogen), CD45 FITC (clone HI30, Becton Dickinson (BD) Pharmingen, Vianen, the Netherlands), HLA-DR PE-Cy7 (clone L243, Sony Biotechnology, Surrey, UK) or with corresponding isotype control antibodies. Staining with HLA -ABC Pe-Cy7 (clone G46-2.6, Biolegend, London, UK) served as a positive control and was used to set-up the correct compensation configuration settings. Cells were measured on a FACS CANTO II (BD) and data were analyzed using FlowJo software 9.9.3 (Tree Star, Ashland, OR).
6
Cell Viability Assay for Breast Cancer
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MRC5 human male foetal lung fibroblasts, SV40 transformed, and MDA-MB-231, human Caucasian female breast adenocarcinoma cells were sourced from the European Collection of Cell Cultures (ECACC) and maintained in Minimal Essential Medium with Glutamax and Earl's salts (MEM, Gibco), supplemented with 10% (v/v) foetal bovine serum (FBS, Pan Biotech) at 37° C in a humidified atmosphere with 5% CO2. Cell passage was performed when cells were 70–80% confluent. Cells were first washed with Dubecco's Phosphate Buffered Saline without calcium chloride or magnesium chloride (DPBS, Sigma Aldrich) prior to the addition of cell dissociation agent, TripLE Select using 1 mLcm−2 (ThermoFisher Scientific). Cell viability was measured with the CellTiter-Blue reagent (Promega) per the manufacturer's instructions. Cells were plated in clear-bottomed 96-well plates at a density of 5000 cells per well. The inhibitors were added the following day, and cell viability was measured 24 hours later using the Synergy HT Multi-Detection Reader (BioTek). Relative cell viability at a given inhibitor concentration was determined by comparing the fluorescence to that of DMSO treated cells.
7
Maintaining Female Human Embryonic Stem Cells
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Human embryonic stem cells (hESCs, female) carrying a GFP reporter for NKX2. ] were maintained on irradiated mouse embryonic fibroblasts (MEF feeder cells) in hESC medium (DMEM/F12-based medium containing 20% knockout serum replacement (ThermoFisher) and 10 ng/mL basic fibroblast growth factor (bFGF) (Miltenyi Biotech)). Passaging of hESCs on MEFs was done by dissociation using 1× TripLE Select (ThermoFisher), subsequent inactivation by dilution in hESC medium, collection of the cells by centrifugation at 240 ×g for three minutes, resuspension of the pellet in 1 mL hESC medium, manual counting using Trypan blue (ThermoFisher) and finally seeding as single cells on freshly prepared plates with MEF cells on 0.1% gelatin.
8
Isolation and Expansion of BM-Derived MSCs
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Isolation of BM-derived MSCs was performed as described previously [29 (link)] through gradient separation and plastic adherence. Briefly, 8 mL of undiluted BM aspirate were loaded into a BD Vacutainer® CPT™ tube (Becton Dickinson, Franklin Lakes, NJ, USA) and then processed according to the manufacturer’s instructions. The top layer containing plasma and mononuclear cells was harvested. The cell number was counted and the viability evaluated. For expansion, cells were then transferred to 150-cm2 culture flasks by seeding 4 × 105 cells/cm2 with α-Modified Minimum Essential medium (α-MEM; BioWhittaker, Lonza, Verviers, Belgium) supplemented with 20% lot-selected fetal bovine serum (FBS; Gibco, Invitrogen-Life Technologies, Paisley, UK) and 1% GlutaMAX™ (Invitrogen-Life Technologies). The flasks were incubated at 37°C in a humidified atmosphere of 5% CO2 with medium change every 3–4 days. When the cells reached ~70–80% confluence, they were detached by mild trypsinization (TripLe™ Select, Invitrogen-Life Technologies) for 3 min at 37°C and counted. Cells were reseeded into a new 150-cm2 flask at a density of 4000 cells/cm2.
9
Immunophenotyping of dissociated cortical cells
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The cortex was dissected into small pieces in cold PBS; these pieces were then incubated with TripLE Select (Invitrogen) at 37 °C for 10 min. After dissociation, cells were immunostained with PBS containing 0.5% BSA and 0.1% NaN3. LIVE-DEAD Fixable Far Red Dead Cell Stain Kit for 633 or 635 nm (Thermo Fisher Scientific) was used for the staining of dead cells. Using Perm & Fix (Invitrogen), the cells were stained using RFP-biotin (Abcam), NeuN-FITC (Millipore) or Tuj1-FITC (eBioscience) (all used at 1:500). Streptavidin-PE (eBioscience) was used at 1:2000. Antibody reaction was done for 30 min on ice. The recording and analysis were performed using BD LSRII with Green laser and DIVA software, and FlowJo (TreeStar), respectively. A total of approximately 100,000 cells were recorded per sample.
10
Isolation and Quantification of mHsp70 in Dissociated Cortical Cells
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The cells were first dissociated with TripLE Select (Invitrogen) from the dissected cortex, and were sorted using FACS Vantage SE. Total RNA was isolated using the RNeasy Plus kit (QIAGEN), and the quality of the total RNA was evaluated by Bioanalyzer RNA 6000 kit (Agilent). Samples showing RNA integrity number >9 were used. cDNA was synthesized by using SuperScript First-strand synthesis system for RT–PCR with random hexamer primers (Invitrogen). GAPDH levels were detected by Taqman rodent GAPDH control reagents and used for normalization. Thermocycling was carried out by using the Applied Biosystems 7900 system and monitored by SYBR Green I dye detection. For qRT–PCR of mHsp70; Forward 5′-ggccagggctggattact-3′ and Reverse 5′-gcaaccaccatgcaagatta-3′ primers were used.
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