PDOs were harvested with TrypLE Express and neutralised with DMEM/F12 Ham medium (Sigma Aldrich) with 10% FBS. Cells were filtered through a 70um filter, counted and re-suspended in phenol-red free RPMI medium (Thermo Fisher) supplemented with 10% FBS (Biosera), 1XGlutamax and 100units penicillin-streptomycin. On day 0, 5000 tumor cells per well of a 96 well-plate (Corning Special Optics Microplate) were plated. CD8 T cells were added on day 1 at the indicated effector to target (E:T) ratios with 20nM of cibisatamab or 20nM of the untargeted negative control antibody DP47-TCB (both provided by Roche). Tumor cells without CD8 T cells and without antibody were also included as controls. All conditions were plated in triplicates and at least 3 different healthy donors were tested on each of the 8 PDOs.
Phenol red free rpmi medium
Manufactured by Thermo Fisher Scientific
Sourced in United States
Phenol red free RPMI medium is a cell culture medium that does not contain the pH indicator dye phenol red. It is designed for use in applications where the presence of phenol red may interfere with assays or imaging techniques.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (2 mentions)
Hepes buffer, by Thermo Fisher Scientific (2 mentions)
β mercaptoethanol, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Dmem f12 ham medium, by Merck Group (1 mentions)
Cibisatamab, by Roche (1 mentions)
Fetal bovine serum (fbs), by Biosera (1 mentions)
Axio observer z1 inverted, by Zeiss (1 mentions)
Csu x1, by Zeiss (1 mentions)
Wavefx x1, by Quorum Technologies (1 mentions)
H2dcfda, by Merck Group (1 mentions)
Synergy 2, by Agilent Technologies (1 mentions)
Penicillin streptomycin glutamine (psg), by Thermo Fisher Scientific (1 mentions)
Tryple select, by Thermo Fisher Scientific (1 mentions)
Ecl advance western blotting detection reagent, by GE Healthcare (1 mentions)
Poly adp ribose polymerase parp, by Cell Signaling Technology (1 mentions)
7 protocols using phenol red free rpmi medium
1
PDO-based Cytotoxicity Assay with Cibisatamab
2
Live cell imaging of Clover-YAP in MDA-MB231 cells
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Live cell imaging of Clover-YAP-expressing MDA-MB231 cells was carried out using a custom WAVE-FX-X1 spinning disc confocal system (Quorum Technologies) with a modified Yokogawa CSU-X1 scanhead on an AxioObserver Z1 inverted microscope (Carl Zeiss) with a ×40 NA 1.2 Plan Apochromat (Carl Zeiss) objective. Cells were plated in a 35 mm glass-bottom dish (Mat-Tek, P35G-1.5-14-C) and were maintained in a stage-top incubator at 37 °C and 5% CO2 during imaging. Cells were cultured in phenol-red free RPMI medium (Thermo Fisher Scientific, 11835030) with 5% FBS and 200 nM SiR-DNA (Spirochrom, SC007) was added 1 h prior to imaging to visualize the nuclei. Localization of Clover-YAP was monitored every 10 min for 2 h. Volocity software was used for image acquisition and processing.
3
Measuring ROS in BMDMs with MSU
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BMDMs were cultured in 96-well plates (1 × 106 cells/well) using phenol red-free RPMI medium (Gibco) for 24 h at 37°C in a humidified incubator with 5% CO2. Cells were then loaded with the ROS-specific fluorescent probe H2DCFDA (20 μM final concentration; Sigma-Aldrich) for 30 min, washed twice with preheated medium, and exposed to MSU (200 μg/ml). In the treatment group, cells were incubated with 10 μM CP105,696 for 40 min prior to the addition of the MSU. Fluorescence was assessed at 10-min intervals over 1 h using a spectrofluorometer (Synergy 2; BioTek) with a fluorescein isothiocyanate filter (excitation 485 nm, emission 538 nm).
4
Isolation and Co-culture of Human Pancreatic Islets and HUVECs
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The use of human pancreatic islets was approved by the Health Research Ethics Committee of the University of Alberta. The all patients or their family’s informed consent was obtained by Dr. T. Kin (Clinical Islet Laboratory, University of Alberta Hospital). Human pancreas donated to the University of Alberta were used to prepare pancreatic islets according to the protocol approved by the Health Research Ethics Committee of the University of Alberta. Isolation of CD82+ cells from islets was carried out at the University of Tokyo according to the guideline of the Life Science Research Ethics and Safety at the University of Tokyo. Upon arrival, islets were incubated for 10 min at 37 °C in 1 × TrypLE select (GIBCO), followed by dissociation of pancreatic islets by pipetting. Cells were isolated using a Moflo cell sorter. HUVEC was added to isolated pancreatic islets at a ratio of 1: 1 and placed in a low attach 96-well M plate (SUMITOMO BAKELITE) at 50,000 cells/well. Phenol red-free RPMI medium (SIGMA) supplemented with 10% FBS (GIBCO) and 1% PSG (GIBCO) was used as a medium. The next day cell clusters were collected for analysis.
5
Cell Culture Optimization for INS-1 and MIN6B1
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INS-1 832/3 cells, were cultured in phenol containing (and 24hours prior to an experiment) phenol red free RPMI medium (Gibco) containing 11mM glucose, supplemented with charcoal stripped FBS (10% v/v; Invitrogen), Sodium Pyruvate (1mM; Gibco), HEPES buffer (10mM; Invitrogen), GlutaMAX (2mM; Gibco), β-mercaptoethanol (50μM; Invitrogen) and, Penicillin-Streptomycin (1x; Gibco). MIN6B1 cells were cultured in DMEM (Invitrogen) containing glucose (4.5 g/L), supplemented with FBS (15% v/v; Invitrogen), β-mercaptoethanol (50μM; Invitrogen) and penicillin/streptomycin (1% v/v; Invitrogen). Cells were seeded in 24-well plates with glass coverslips using medium containing charcoal-stripped FBS (10% v/v). Both cell types were cultured at 37°C in a humidified incubator containing 5% CO2.
6
Cell Culture Optimization for INS-1 and MIN6B1
7
Cell Viability Assay and Western Blot Analysis
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The Ez-Cytox cell viability assay kit was obtained from the Daeil Lab Service Co. (Seoul, Korea). RIPA buffer, primary antibodies against BH3-interacting domain (BID), Bax, Bcl-2, cleaved caspase-8, cleaved caspase-3, cleaved caspase-9, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), poly ADP ribose polymerase (PARP), and horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibodies were obtained from Cell Signaling (Danvers, MA, USA). The Pierce™ BCA Protein Assay Kit was obtained from Thermo Scientific (Waltham, MA, USA). RPMI1640 medium was purchased from Cellgro (Manassas, VA, USA). Fetal bovine serum (FBS) and phenol-red-free RPMI medium were obtained from Gibco BRL (Grand Island, NY, USA). Charcoal-dextran-stripped human serum was purchased from Innovative Research (Novi, MI, USA). ECL Advance Western blotting detection reagents were obtained from GE Healthcare (Little Chalfont, UK).
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