Human embryonic kidney 293T (HEK293T) cell lines (from embryonic kidney of female human fetus) and Vero cell lines (from the kidney of a female normal adult African green monkey) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, C11995500BT) supplemented with 10% fetal bovine serum (FBS) (Gibco, 10270-106). A549 human lung adenocarcinoma cell lines (from the lung of a 58 years old male human) and DLD1 human colorectal adenocarcinoma cell lines (from the colorectum of an adult) were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, C11875500BT). HCT116 human colorectal adenocarcinoma cell lines (from the colorectum of a male human) were cultured in McCoy’s 5A medium (KeyGEN, KGM4892N-500). All cell lines were cultured at 37°C under 5% CO2. 293T (#CRL-11268), Vero (#CCL-81), A549 (#CCL-185), DLD1 (#CCL-221), and HCT116 (#CCL-247) were originally obtained from ATCC and all human cell lines were authenticated by China Center for Type Culture Collection (CCTCC). Mycoplasma contamination was routinely checked by PCR analysis and eliminated by treatment with Plasmocin™ (ant-mpt). The primers were as follows: Myco forward 5′-GGG AGC AAA CAG GAT TAG ATA CCC T-3′; Myco reverse 5′-GCA CCA TCT GTC ACT CTG TTA ACC TC-3′.
C11875500bt
Manufactured by Thermo Fisher Scientific
Sourced in United States
The C11875500BT is a laboratory equipment product from Thermo Fisher Scientific. It is designed for use in scientific research and analysis. The core function of this product is to provide a specific set of capabilities to support laboratory workflows, however, a detailed description cannot be provided while maintaining an unbiased and factual approach.
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Lab products found in correlation
C11995500bt, by Thermo Fisher Scientific (15 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (5 mentions)
Rpmi 1640, by Thermo Fisher Scientific (4 mentions)
Dnase 1, by Roche (3 mentions)
Liberase tl, by Roche (2 mentions)
Trypan blue, by Thermo Fisher Scientific (2 mentions)
Hek293t, by Thermo Fisher Scientific (2 mentions)
Beas 2b, by Thermo Fisher Scientific (2 mentions)
M csf, by Thermo Fisher Scientific (2 mentions)
C8050, by Solarbio (1 mentions)
Hct15, by American Type Culture Collection (1 mentions)
Dld 1 ccl 221, by American Type Culture Collection (1 mentions)
Ccd 18co, by American Type Culture Collection (1 mentions)
Caco 2 htb 37, by American Type Culture Collection (1 mentions)
Fnd500, by ExCell Bio (1 mentions)
Hct 8 ccl 244, by American Type Culture Collection (1 mentions)
Counting chamber, by Brandel Inc (1 mentions)
Hiseq x10, by Illumina (1 mentions)
Begm medium, by Lonza (1 mentions)
42 protocols using c11875500bt
1
Culturing Human and Primate Cell Lines
2
Harvesting Peritoneal Macrophages
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We harvested the peritoneal macrophages by injecting and shaking 14 mL (7 mL × 2) of ice bath washing buffer containing RPMI-1640 (C11875500BT, Gibco, Thermo Fisher Scientific) and 1% heat-inactivated fetal bovine serum (FBS; 42Q6395K, Gibco, Thermo Fisher Scientific). Collected cells were centrifuged at 1250g for 3 minutes at 4°C. Cells were resuspended in 2 mL of culture medium and seeded in a culturing plate. To purify the macrophages, the cell culture medium was changed after seeding in a humidified atmosphere containing 5% CO2.
3
EHEC O157:H7 Strain Manipulation and Characterization
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The EHEC O157:H7 EDL 933 (WT) and its isogenic strains lacking the espF gene (ΔespF, with kanamycin resistance), complemented strain (ΔespF/pespF, with kanamycin and chloramphenicol resistance) were constructed in the previous work (Wang et al., 2017 (link)). The espF gene expression of the complemented strain was induced by L -arabinose. The bacterial strains were grown in Luria Bertani media (Oxiod #LP0137, Basingstoke, United Kingdom) at 37°C at 200 rpm in a constant-temperature, oscillating shaker with appropriate antibiotics: 100 μg/ml kanamycin (Solarbio #K8020, Beijing, China) for ΔespF and ΔespF/pespF; 10 μg/ml chloramphenicol (Solarbio #C8050, Beijing, China) and 2 mg/ml L -arabinose (Solarbio #L8060, Beijing, China) for ΔespF/pespF strain.
HT-29, Vero, Hela and Caco-2 cells were preserved in our laboratory. Briefly, they were grown in RPMI-1640 (Gibco #C11875500BT, New York, NY, United States) or DMEM medium (Gibco # C11995500BT, New York, NY, United States) containing 10% fetal bovine serum (Gibco #10270-106, New York, NY, United States) and 1% penicillin/streptomycin (Gibco #15140122, New York, NY, United States). The cells were cultured in tissue culture plates at 37°C under humidified 5% CO2 prior to infection.
HT-29, Vero, Hela and Caco-2 cells were preserved in our laboratory. Briefly, they were grown in RPMI-1640 (Gibco #C11875500BT, New York, NY, United States) or DMEM medium (Gibco # C11995500BT, New York, NY, United States) containing 10% fetal bovine serum (Gibco #10270-106, New York, NY, United States) and 1% penicillin/streptomycin (Gibco #15140122, New York, NY, United States). The cells were cultured in tissue culture plates at 37°C under humidified 5% CO2 prior to infection.
4
Immortalized Breast Cell Lines for Tumor Microenvironment Research
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CAFs and NFs were isolated from breast tumor tissues and their paired normal tissues and identified with CAF-related biomarkers, respectively; and were immortalized by human telomerase reverse transcriptase gene (hTERT) as described previously [16 ,25 (link)]. NFs and CAFs were cultured in DMEM (C11995500BT, Gibco) with 10% FBS (10099-144, Gibco, Austrlia); Human breast cancer cells MDA-MB-231 and BT-549 were maintained in RPMI-1640 medium (C11875500BT, Gibco) with 10% FBS at 37 °C in a tri-gas incubator containing 5% CO2 under normoxic (21% O2) or hypoxic (1% O2 and 94% N2) condition. The genotyping of these breast cancer cells shared >94% of the STR loci with the ATCC original clones by STR identification (Genewiz, Suzhou).
5
Human Colorectal Cell Lines Protocol
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The human CRC cell lines LS174T (CL-188), RKO (CRL-2577), DLD-1 (CCL-221), Caco2 (HTB-37), SW620 (CCL-227), HCT-8 (CCL-244), HCT116 (CCL-247), and HCT-15 (CCL-225); the human normal colorectal epithelial cell line FHC (CRL-1831); and the human normal colorectal fibroblast cell line CCD-18Co (CRL-1459) were all purchased from the American Type Culture Collection (ATCC). All human CRC cells were cultured in RPMI-1640 medium (Gibco, C11875500BT) supplemented with 10% fetal bovine serum (FBS) (ExCell Bio, FND500). FHC was cultured in RPMI-1640 medium supplemented with 15% FBS, and CCD-18Co was cultured in Eagle’s Minimum Essential Medium (EMEM) (ATCC, 30–2003) supplemented with 10% FBS. All cells were cultured at 37 °C in humidified atmosphere containing 5% CO2. Cell line certificates of analysis were obtained from the ATCC. All cell lines were negative for mycoplasma.
6
Isolation of Human Fetal Adrenal and Gonad Cells
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Fresh samples of human fetal adrenal glands or gonads were placed in RPMI 1640 medium (C11875500BT, Gibco), which contained 5% fetal bovine serum (FBS; SH30070.03, HyClone) and 1% penicillin–streptomycin (15140-122, Gibco), and quickly transported to the laboratory on ice. Then, the fetal adrenal glands and gonads were separated under a stereomicroscope (Nikon), washed with cold D-PBS (311-425-CL, Wisent) and sliced into approximately 1–2-mm3 pieces. The tissues were transferred to digestion solution [0.1 mg/ml Liberase TL (5401020001, Roche) and 1 mg/ml DNase I (10104159001, Roche) in RPMI 1640] at 37°C with gentle shaking throughout (adrenal glands for 20 min and gonads for 30 min), filtered through a 100-μm cell strainer (352360, Falcon), centrifuged and resuspended in 5 ml of 1X red blood cell (RBC) lysis buffer (420301, BioLegend) for 5 min on ice. Then, the cells were washed twice with D-PBS containing 1% FBS, filtered through a 40-μm cell strainer (352340, Falcon), centrifuged and resuspended in DPBS with 1% FBS. The cell number and viability were assessed by Trypan blue (152 50-061, Gibco) staining and counting in a counting chamber (717805, Brand).
7
Single-cell Sequencing of Kidney Clear Cell Carcinoma
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Single-cell transcriptome sequencing data (GSE152938) was downloaded form GEO database. Prior literature has outlined the steps used to prepare single-cell suspensions (23 (link)). In brief, cold Hank’s Balanced Salt Solution was utilized to transport freshly isolated tumor samples from the operating room to the lab (HBSS; Gibco, C11875500BT). Afterward, the samples were rinsed and sliced into 2-4 mm sections. For 30 minutes, several species of tissue were gently agitated in a digesting solution comprised of HBSS at 37°C. Before single-cell sequencing, samples were washed and filtered to remove red blood cells and determine cell viability. Two samples of kidney clear cell carcinoma (KIRC) were obtained from patients who underwent radical nephrectomy. Hiseq X10 (Illumina, San Diego, California) with standard settings was utilized to sequence all the samples. CellRanger (v3.0.2) was utilized to transform preliminary sequencing data (.bcl) into FASTQ files. To perform quality control (QC) and secondary analysis, we employed the R programming language (v3.5.2) together with the Seurat R package (v3.1.1). The GEO database (GSE152938) contains the datasets derived by single-cell sequencing (24 (link)).
8
Cell Line Cultivation Protocol
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Human LA NCI-H1975 cell line (20180612-06, OBIO, Shanghai) and human bronchial epithelial cell (BEAS-2B) were purchased from OBiO Technology (Shanghai) Corp., Ltd. ((Shanghai, China).Shanghai, China). NCI-H1975 cells were cultured in 1640 medium (C11875500BT, Gibco, Thermo Fisher Scientific, Inc.), supplemented with 10% fetal bovine serum (FBS)(10270-106, GIBCO), 1μg/mL puromycin. The cell lines were incubated at 37 °C with 5% CO2. BEAS-2B cultures were maintained in BEGM medium (Lonza, Cobioer). All cells were cultured at 37 °C with 5% CO2.
9
Maternal PBMC Therapy for Early Pregnancy Loss
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Female mice were randomly divided on Pd0.5 into three groups: control group, EID group, and EID with PBMCs group. On Pd1.5, PBMCs were isolated from total blood of non-pregnant female mice (see below for detailed steps) and washed with phosphate buffered saline (PBS) buffer. The washed PBMCs were resuspended in basal culture medium without fetal bovine serum (FBS) or fetal calf serum (FCS). Then the PBMCs (1–2 × 106 in 2.5 μl) were gently injected surgically into the bilateral uterine horn of mice in the EID with PBMCs group using a microsyringe. The mice in the other groups received the same amount of basal culture medium (Roswell Park Memorial Institute 1640 (RPMI 1640; C11875500BT, Gibco®, Waltham, MA, USA). The EID and the EID with PBMCs groups were obtained by injecting the mice with 0.08 mg mifepristone (H10950003, Zizhu Pharmaceutical, Beijing, China) subcutaneously at 0900 h on Pd3.5.
10
Cultivation of Ovarian and Lung Cancer Cell Lines
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A2780 and A2780T human ovarian cancer cell lines and the A549 lung cancer cell line were obtained from Type Culture Collection of Shanghai Meixuan Biotechnology Co., Ltd (cat. nos. MXC020, MXC021 and MXC026). All cells were cultured in RPMI 1640 medium (Gibco, C11875500BT) at 37°C in a 5% CO2 humidified incubator. All media contained 10% FBS(AusGeneX, LV-FBSCN500S), 100 U/ml penicillin and 100 µg/ml streptomycin (Beyotime, C0222). A2780T cells were cultured in a drug-free medium for 7 days to avoid the interference of drug toxicity on the experimental results. Logarithmic growth phase cells were taken for the experiments.
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