Tumor and blood samples were collected simultaneously during surgery. Tumor samples were mechanically dissociated on ice with 10% fetal bovine serum-phosphate-buffered saline (FBS-PBS), filtered using a 70-micron strainer, and washed with 10% FBS-PBS. All blood samples were collected using a collection tube containing ethylenediaminetetraacetic acid-2Na. For both the tumor and blood samples, mononuclear cell components were separated using density-gradient centrifugation with a Ficoll-Paque PLUS (Cytiva Inc., Tokyo, Japan) according to the manufacturer’s instructions. Then, they were suspended in a CELLBANKER I (Takara Bio Inc. Shiga, Japan) and stored in liquid nitrogen.
Cellbanker 1
Manufactured by Takara Bio
Sourced in Japan
CELLBANKER 1 is a serum-free cryopreservation medium designed for the long-term storage of cells. It is formulated to maintain cell viability and functionality during the freezing and thawing process.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (2 mentions)
Ficoll paque plus, by Cytiva (1 mentions)
Histopaque 1077, by Merck Group (1 mentions)
Qiashredder, by Qiagen (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Ficoll paque density gradient centrifugation, by GE Healthcare (1 mentions)
Rnalater stabilization solution, by Thermo Fisher Scientific (1 mentions)
Jetprime, by Polyplus Transfection (1 mentions)
Neon transfection system, by Thermo Fisher Scientific (1 mentions)
Kod fx neo, by Toyobo (1 mentions)
Gentlemacs, by Roche (1 mentions)
Collagenase d, by Roche (1 mentions)
Gentlemacs, by Miltenyi Biotec (1 mentions)
Af594 ib4, by Thermo Fisher Scientific (1 mentions)
Phosphatase inhibitor cocktail, by Nacalai Tesque (1 mentions)
Hexadimethrine bromide, by Merck Group (1 mentions)
Anti phospho vegfr2 tyr1175, by Cell Signaling Technology (1 mentions)
Anti vegfr2 antibody, by Cell Signaling Technology (1 mentions)
Ly294002, by Nacalai Tesque (1 mentions)
Recombinant human fgf basic b fgf, by Thermo Fisher Scientific (1 mentions)
12 protocols using cellbanker 1
1
Simultaneous Tumor and Blood Sample Collection
2
Isolation and Cryopreservation of PBMCs
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Peripheral blood was collected at baseline, Cycle 3 Day 1 (C3D1), C5D1, C9D1, and on progression disease (PD) (when available), and then centrifuged using Histopaque-1077 (Sigma Aldrich, St Louis, MO, USA) as previously described. Briefly, the samples were diluted with PBS and layered on Histoaque-1077 before being centrifuged at 800 × g for 15 min at room temperature. Mononuclear cell components were collected, washed twice with PBS, and stored at -80°C in CELLBANKER I (Takara Bio Inc. Shiga, Japan). These PBMCs were then frozen in liquid nitrogen until use.
3
Tamoxifen Exposure in Cell Cultures
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Cells were exposed or unexposed to tamoxifen for the indicated time periods in sextuplicate. Control cells from the 1st week to the 12th week and tamoxifen-treated cells of the 1st week and from the 5th week to the 12th week were re-plated at a density of 2 × 106 cells/100-mm dish and grown for 2 days to reach the log phase (~50% confluent). For the 2-, 3-, and 4-week tamoxifen-treated cells, the medium was changed instead of re-plating the cells, and the cultures were grown for 2 days. Therefore, the RNA samples from the 2-, 3-, and 4-week tamoxifen-treated cells were prepared from cultures in log phase but at a different confluency (20%–80% confluent). The tamoxifen exposure period was defined as up to the day of sample preparation. Quintuplicate samples (quadruplicate for the 2-week control sample) were used for total RNA extraction with Qiashredder (QIAGEN) and an RNeasy mini kit (QIAGEN). RNA concentration and integrity was evaluated using a Bioanalyzer 2100 (Agilent). For the validation study, the remaining cells were trypsinized, re-suspended in Cell Banker I (TaKaRa) freezing medium, and frozen in liquid nitrogen.
4
Isolation and Characterization of VZV Strain
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The VZV wildtype strain 1710, which was isolated in our laboratory from the
blister contents of a 9-year-old girl with HZ, was used in this study. MeWo
cells that were infected with VZV were collected and frozen using CELLBANKER1
(Takara Bio) until the experiments were conducted. For the infection
experiments, the cells were infected with the frozen infected cells after
thawing. The titers of infected cells were determined using a plaque assay of
frozen infected cells after thawing on MeWo cells.
blister contents of a 9-year-old girl with HZ, was used in this study. MeWo
cells that were infected with VZV were collected and frozen using CELLBANKER1
(Takara Bio) until the experiments were conducted. For the infection
experiments, the cells were infected with the frozen infected cells after
thawing. The titers of infected cells were determined using a plaque assay of
frozen infected cells after thawing on MeWo cells.
5
Bronchoalveolar Lavage Sampling Protocol
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BAL was performed following the standard protocol, which involved injecting three 50 mL volumes of normal saline (150 mL in total) into the wedged segmental bronchus leading to the target tumor lesion that was assessed radiologically (Supplemental Figure 2 , B–E). Blood and BALF were collected in tubes on the same date, and plasma and BALF supernatants were collected after centrifugation and stored at −80°C for analysis. Cells in BALF were stored in RNAlater Stabilization Solution (Thermo Fisher Scientific) for RNA sequencing. PBMCs were isolated using Ficoll-Paque density gradient centrifugation (GE Healthcare) and stored in CELLBANKER 1 (Takara Bio) at −80ºC for analysis.
6
ATAC-seq analysis of AZD9291 response
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HCC827 cells were treated with DMSO or AZD9291 (1 mM) in replicates for 18 h. The cells (3 × 105) were then frozen in cell stock solution (Cell Banker 1, TAKARA) and transported to DNAFORM Life Science Research Center 402 (Yokohama, Japan) to perform ATAC-seq. Next-generation sequencing was performed using 150 bp pair-end sequences at 40 million reads/sample. The quality of the FASTQ sequences was determined using Fast QC. The quality-filtered sequences were mapped to the reference genome hg38 using BWA. BAM files were used for peak calls with PePr, in which the replicate peaks were merged. The detected peaks were annotated using HOMER. The motifs within the annotated peaks were searched using FIMO (http://meme-suite.org/tools/fimo ). The significantly increased open chromatin peaks in the AZD9291-treated group were visualized using the IGV genomic browser.
7
Efficient Primary Cell Culture for SCNT
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The primary cell culture using cells from tail tip tissues were performed as described previously [22 , 23 (link)]. Briefly, the tail tip tissues from B6D2F1 mice (aged 8–10 weeks) and from large Japanese field mice were obtained from anesthetized animals. The cells were incubated
in DMEM medium supplemented with 10% fetal bovine serum and 2.5 µg/ml amphotericin B to allow for cell migration at 37°C under 5% CO2 in air. Cells undergoing migration
were used for passaged culture or stocked after freezing using a CELLBANKER 1 (Takara Bio, Shiga, Japan; CB011). As donor cells, the tail tip cells derived from laboratory mice
were used at passages 3 to 5, and the tail tip cells derived from large Japanese field mice were used at passages 8 to 14. The preparation of donor cells for SCNT or iSCNT were
performed as previously described [20 (link), 21 ]. Briefly, the cell pellets were re-suspended in Hepes-CZB
(HCZB) medium supplemented with 6% d-BSA and placed on ice until cell fusion.
in DMEM medium supplemented with 10% fetal bovine serum and 2.5 µg/ml amphotericin B to allow for cell migration at 37°C under 5% CO2 in air. Cells undergoing migration
were used for passaged culture or stocked after freezing using a CELLBANKER 1 (Takara Bio, Shiga, Japan; CB011). As donor cells, the tail tip cells derived from laboratory mice
were used at passages 3 to 5, and the tail tip cells derived from large Japanese field mice were used at passages 8 to 14. The preparation of donor cells for SCNT or iSCNT were
performed as previously described [20 (link), 21 ]. Briefly, the cell pellets were re-suspended in Hepes-CZB
(HCZB) medium supplemented with 6% d-BSA and placed on ice until cell fusion.
8
Stable Transfection of Mouse ESCs
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mESCs were cultured in six-well plates to ~ 50% confluence and transfected with 2 μg of plasmid DNA using 4 μL of jetPRIME (Polyplus) per well. For the establishment of mESCs stably expressing 3 × FLAG-H2A3, cells were replated onto a 10-cm dish at 24 h after transfection and cultured for a week in the presence of 10 μg/mL puromycin. Puromycin-resistant colonies were transferred individually to a 96-well plate. Cells that expressed the fusion proteins were expanded, collected, frozen in liquid nitrogen, and stored in cell cryopreservation medium (CELLBANKER 1, Takara Bio) at − 80 °C.
9
CRISPR-Cas9 Knockout of Pluripotency Genes
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Before transfection, plasmid DNA encoding Cas9 and antibiotic resistance genes (blasticidin or neomycin) was digested with PmeI (New England BioLabs). Each digested plasmid DNA (1 µg fragment each containing blasticidin and neomycin resistance genes) and 1 µg pX330 plasmid coding gRNA targeting the 3′ untranslated region or downstream of NANOG, SOX2, or POU5F1 were transfected into the suspended DU145 (2.6 × 105 cells/40 μL) using the Neon Transfection System (Invitrogen) (voltage: 1000, width: 30; pulse number: 2), and cultured in a 10 cm dish. After 2 days of transfection, blasticidin (7.5 μg/mL) and G418 (150 μg/mL) were added to a 10 cm dish. Single colonies were selected and individually cultured in 96-well plates followed by 24-well plates. Half of the sub-confluent cells were cryopreserved in CELLBANKER 1 (Takara Bio). The remaining cells were used to extract genomic DNA by heating at 95 °C for 10 min in the presence of 50 mM NaOH followed by the addition of 0.1% volume of 1 M Tris–HCl (pH 8.0) for neutralization. Genotyping PCR was performed using KOD FX Neo (TOYOBO).
10
Comprehensive Immune Profiling Protocol
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This study was conducted as described in the Ethics Statement. All samples were collected at Nagoya City University Hospital. Samples were prepared as previously reported for T cells5 and DCs.13 , 14 , 15 Briefly, some parts of the samples were embedded in O.C.T. Compound (Tissue Tek, Sakura Fintek), frozen, and stored at −80°C until use. Other parts of the samples were mechanically crushed using Gentle MACS (Miltenyi Biotec). Some samples used to analyze DCs were treated with collagenase D (Roche) before using Gentle MACS. After washing, the cells were frozen in CELLBANKER 1 (Takara Bio) and stored in liquid nitrogen. Separate analyses of bulk RNA sequencing (RNA‐Seq) and flow cytometry to identify surface‐CTLA‐4+ Treg cells have been previously published.5 All flow and mass cytometry analyses in this study are novel and unpublished. The characteristics of the patients are listed in Table S1 .
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