To allow the cells to recover from the detrimental effects of cryopreservation 5 , MSCs were thawed in a 37°C water bath, diluted 10‐fold with prewarmed 37°C D4 culture media and washed once before being placed into tissue culture in D4 culture media at a target density of 2,000–3,000 cells per cm2 (range, 1,000–5,000). This culture will always be planned for a 7‐ or 9‐day culture prior to a patient's scheduled infusion. Two days before the expansion was complete, cells were fed with fresh D4 culture media containing 500 U/ml GMP‐grade recombinant human IFNγ (R&D Systems, Minneapolis, MN, http://www.rndsystems.com ). A sample of spent media from each culture vessel was collected 2 days prior to completion and tested for microbial contamination to satisfy the appropriate release criteria. On the day of the planned infusion, the cells were collected by trypsinization (Gibco), washed to remove IFNγ and then resuspended in infusion media (Plasma‐Lyte A [USP], 0.22% NaCl [USP], 2.5% Dextrose [USP], 0.5% human serum albumin [USP]). This final cell product or spent media was tested and met all release criteria which will be required in clinical trials before transport to the subject's bedside.
Trypsinization
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
Trypsinization is a laboratory technique used to detach adherent cells from a culture surface. Trypsin, a proteolytic enzyme, is used to cleave the cell-surface proteins that anchor the cells to the substrate, enabling their suspension and subsequent use in downstream applications.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (8 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (7 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (4 mentions)
Facscalibur flow cytometer, by BD (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
L glutamine, by Thermo Fisher Scientific (3 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (3 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Colcemid, by Thermo Fisher Scientific (2 mentions)
Isis software, by MetaSystems (2 mentions)
Vectashield antifade medium, by Vector Laboratories (2 mentions)
β mercaptoethanol, by Merck Group (2 mentions)
Hepes, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin glutamine (psg), by Thermo Fisher Scientific (2 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (2 mentions)
Pen strep, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
61 protocols using trypsinization
1
Thawing and Expansion of Cryopreserved MSCs
2
Quantifying MBNs-NH2 Internalization in rDPSCs
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To quantify the internalization of MBNs-NH2 into the cells and to determine the pathway of internalization, 150 μg of FITC-conjugated MBNs-NH2 was incubated with 3×105 rDPSCs in each well of a 6-well plate (50 μg per mL of 105 rDPSCs) for a pre-determined period of time (0.25, 0.5, 1, 2, 4 hours). To determine the pathway of internalization, we pre-incubated rDPSCs with 1 mM of amantadine hydrochloride (A1260, Sigma, St. Louis, MO, USA), 100 mM of genistein (G6649, Sigma), and 2.5 mM of 5-(N-ethyl-N-isopropyl) amiloride (A3085, Sigma) for 1 hour before treating the FITC-conjugated MBNs-NH2. To determine whether the endocytosis was ATP-dependent, the cells were incubated at 4°C for 4 hours or were pre-treated with 100 mM of sodium azide for 1 hour. After incubating with various concentration of each chemical for 6 hours, maximum concertation which showed non-cytotoxicity to rDPSCs was chosen to pretreat the cells. After trypsinization (Gibco), the cells were centrifuged at 10,000 rpm for 5 minutes, and the pellets were fixed with ice-cold PBS. Subsequently, 400 μL of binding buffer was added to each tube prior to analysis with a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). Data for 10,000 cells in each sample were analyzed using the CellQuest Pro software (v.5.1 BD Biosciences).
3
Subcutaneous Tumor Growth Monitoring
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Tumor cells were collected by trypsinization (Gibco) and washed three times with 1× PBS (Gibco). Cells were resuspended in PBS, and 1 × 106 tumor cells were injected subcutaneously into the flanks of mice. Subcutaneous tumor area measurements (calculated as length × width) were collected 2–3 times a week using digital calipers until the endpoint of the study.
4
Discriminating Cell Populations by Annexin-V/PI Staining
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Annexin-V/PI double staining can discriminate necrotic, apoptotic, and viable cells population by flowcytometry. Annexin V-FITC/PI (Sigma-Aldrich kit, USA) was used according to the manufacturer's protocol. Equal numbers of MCF7 (1 × 106 cells/ml) were seeded in each well of a six-well plates, allowed to attach, and synchronized. Cells were then cultured in the IC50 value of ATO, CM (100%, 50%), and CM + ATO combination for 48 h. Untreated cells were considered as control groups. Each independent replication performed in triplicate. The cells harvested by trypsinization (Gibco, UK), were washed twice with precooled (4 °C) PBS, incubated with 5 μl Annexin V-Fluorescein isothiocyanate for 15 min, followed by incubation with 10 μl Propidium Iodide (PI) for 5 min in dark at room temperature. The number of viable/dead cells and DNA content (during a mitotic cell cycle) were measured by the BD Accuri™ C6 flow cytometer and analyzed with Flow-Jo (version7) software.
5
KRAS Mutant Colorectal Cell Lines
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The colorectal cancer cell lines in this study, including LS411N (colorectal carcinoma), DLD1 (colon adenocarcinoma), RKO (colon carcinoma), HCT116 (colon adenocarcinoma), SW620 (Caucasian colon adenocarcinoma), HT29 (Caucasian colon adenocarcinoma), and SW480 (colon adenocarcinoma), were purchased from American Type Culture Collection (ATCC), and all cell lines are KRAS mutants. All cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (HyClone) supplemented with 10% fetal bovine serum (FBS) (HyClone), 1% penicillin (100 units/mL), and streptomycin (100 μg/mL) (Sigma). These cells were placed in a cell culture incubator supplied with 5% CO2, maintained at 37°C, and passaged at ≥80% confluence by trypsinization (Gibco). All experiments were performed when the cells were in the logarithmic growth phase.
6
Cultivation of CHO-K1 Cell Line
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CHO-K1 cells (BCRC No. CCL-61) purchased from the Culture Collection and Research Center of the Food Industry Institute (Hsin-Chiu City, Taiwan) were maintained in growth medium composed of F-12K supplemented with 10% fetal bovine serum. Cells were subcultured once every 3 days by trypsinization (GIBCO-BRL Life Technologies, Gaithersburg, MD, USA), and the medium was changed every 2–3 days.
7
Mesenchymal Stem Cell Immunophenotyping
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The MSC-like cells were harvested by trypsinization (Gibco) and incubated with 1% bovine serum albumin (BSA) for 1 h at 4 °C to block nonspecific Fc-mediated interactions, then incubated in the dark at 4 °C for 30 min with 400 μl of CD45-FITC (BD Biosciences, 340664, Franklin Lakes, NJ), CD34-APC (BD, 555824), CD105-FITC (BD, 561443), CD73-PE (BD, 561258), and CD90-APC (BD, 559869) antibodies. The cells were stained with PE- or FITC-labeled IgG as an isotype control. The cells were evaluated by a FACSCalibur flow cytometer (Becton–Dickinson, San Jose, CA) and analyzed with FlowJo software (Tree Star Inc., Ashland, Oregon, USA). The percentage of stained cells was calculated relative to the isotype control.
8
Measuring Tumor Cell Migration Inhibition
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Alteration of tumor cell migration induced by UTI and CUR was estimated by an in vitro wounding healing assay. Briefly, log-phase cells were collected after trypsinization (Gibco, Life Technologies) and inoculated into a six-well plate at 5 × 105 (link) cells per well. The plates were cultured in DMEM containing 10% FBS for 24 hours to ~100% confluency in a monolayer. The growth medium was then removed and the wells were cross-scratched with a 200 μL yellow pipette tip and washed three times with serum-free DMEM to remove the cell debris. Cells were incubated with 800 U UTI, 10 μM CUR, 800 U UTI plus 10 μM CUR, and phosphate buffered saline (PBS), respectively, for 0 hours or 24 hours and 48 hours and inspected under an inverted microscope. The distance from one side of the scratch to the other was measured at different intervals using Image Pro-Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).
9
Chondrocyte Isolation and L-Theanine Treatment
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Primary chondrocytes were isolated from 14- to 21-day-old rats. After trypsinization (0.25%, GIBCO, New York, USA) of cartilage for 30 min, the slice was treated with 0.2% collagenase II for 4 h at 37 °C. The culture medium was then cultured with DMEM/F12, including 10% fetal calf serum (Biological Industries, Israel). The steps were consistent with previous research [24 (link)] and the second passage cells were chosen for the subsequent experiments. Identification of chondrocytes was stained with toluidine blue for proteoglycans and immunohistochemical staining for type II collagen (Novus Biologicals, CO, USA).
The medium was cultured with DMEM/F12 containing 0.5% serum starving for 12 h and were stimulated with IL-1β (PeproTech Inc. USA) for 24 h prior to being co-cultured with different concentrations of L-theanine (50, 100, 200 μM) for 24 h. Cells treated with IL-1β only served as the control. The dose of L-theanine (≥98% (high-performance liquid chromatography, HPLC), Sigma-Aldrich, St. Louis, MO, USA) was determined according to previous studies which L-theanine showed inhibition of NF-κB [25 (link)] and anti-inflammation activities [26 (link)].
The medium was cultured with DMEM/F12 containing 0.5% serum starving for 12 h and were stimulated with IL-1β (PeproTech Inc. USA) for 24 h prior to being co-cultured with different concentrations of L-theanine (50, 100, 200 μM) for 24 h. Cells treated with IL-1β only served as the control. The dose of L-theanine (≥98% (high-performance liquid chromatography, HPLC), Sigma-Aldrich, St. Louis, MO, USA) was determined according to previous studies which L-theanine showed inhibition of NF-κB [25 (link)] and anti-inflammation activities [26 (link)].
10
Quantifying ZIKV-Infected Cells by FACS
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To quantify ZIKV-infected cells, cells were rinsed in PBS and dissociated by trypsinization (Gibco, by Life Technologies). Intracellular staining of envelope protein E was performed as previously described (Hubert et al., 2019 (link)). Briefly, detached and dissociated cells were fixated in a 4% paraformaldehyde (PFA) solution for 15 min at RT and permeabilized with PBS – 0.2% Triton for 5 min at RT. Then, ZIKV envelope protein (E) staining was performed using a primary mouse anti-E antibody (4G2) for 20 min at RT, and a secondary Alexa Fluor 488-coupled goat anti-mouse antibody (Life technologies) for 20 min at RT. Data were acquired by fluorescence activating cell sorting (FACS) using Gallios and CytoFLEX Beckman Coulter cytometers, and analysis was performed using FlowJo 10.0.8r1 software.
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