Human normal breast epithelial cells MCF-10A and BC cell lines (MCF7, BT474, SKBr-3, ZR-75-30, MDA-MB-453 and MDA-MB-231) were bought from ATCC (Rockville, USA). These cells were grown in the RPMI-1640 complete medium (Thermo Scientific Hyclone, Utah, USA) which contained 10% fetal bovine serum (FBS, Thermo Scientific Hyclone, Utah, USA) and 1% penicillin/streptomycin (Yeasen Biotech Co., Ltd., Shanghai, China) and kept at 37°C with 5% CO2 and saturated humidity. The medium was replaced every 2 to 3 days. When the cells got 90% confluency, 0.25% trypsin (Thermo Scientific Hyclone, Utah, USA) was adopted for cell trypsinization and subculture.
Complete rpmi 1640 medium
Manufactured by Thermo Fisher Scientific
Sourced in United States, China, Canada, Switzerland
Complete RPMI 1640 medium is a cell culture medium formulated to support the growth and maintenance of a variety of cell lines. It provides essential nutrients, vitamins, and other components required for cell proliferation and survival in vitro.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (56 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (31 mentions)
Streptomycin, by Thermo Fisher Scientific (23 mentions)
Penicillin, by Thermo Fisher Scientific (21 mentions)
L glutamine, by Thermo Fisher Scientific (14 mentions)
Trypsin, by Thermo Fisher Scientific (10 mentions)
Complete dmem medium, by Thermo Fisher Scientific (8 mentions)
Mda mb 231, by American Type Culture Collection (7 mentions)
Glutamax, by Thermo Fisher Scientific (7 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (6 mentions)
Penicillin, by Merck Group (6 mentions)
Lipopolysaccharides (lps), by Merck Group (5 mentions)
Mcf 7, by American Type Culture Collection (5 mentions)
Zr 75 30, by American Type Culture Collection (4 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (4 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Bt474, by American Type Culture Collection (4 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (4 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (4 mentions)
Gm csf, by Thermo Fisher Scientific (4 mentions)
252 protocols using complete rpmi 1640 medium
1
Breast Cancer Cell Culture Protocol
2
Investigating miR-373-3p and TFAP4 in HCC
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The European Collection of Cell Culture (ECACC, Salisbury, UK) supplied us with the normal human thyroid epithelial cell line L-02, while HCC cells (Huh7, HLE, HCCLM6, HCCLM3) were ordered from the College of Science, Institute of Cell Research, Chinese Academy of Sciences (Shanghai, China). The cells were cultivated with an RPMI-1640 complete medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Thermo Scientific Hyclone, Utah, USA) in an incubator (37°C, 5% CO2). The experiment was launched when the cells covered about 90% of the bottle bottom.
Huh7 and HCCLM3 cells underwent 0.25% trypsinization and were seeded on 6-well plates (1 × 105 cells per well). The oligonucleotides of miR-373-3p mimics and their nonspecific controls were bought from RiboBio (Guangzhou, China). Lentiviral vectors incorporating miR-373-3p (Lv-miR-373-3p) were synthesized in cells to represent miR-373-3p overexpression. When the cells achieved 70%–80% fusion, Lipofectamine® 3000 (Life Technologies, San Diego, CA, USA) was utilized to transfect Lv-miR-373-3p and the nonspecific control (Lv-NC), TFAP4 overexpression plasmids (TFAP4), small interfering RNA against TFAP4 (si-TFAP4), si-IGF1, and the corresponding negative control (vector, or si-NC) were transfected into the cells. All transfection steps were conducted as instructed by the supplier [19 (link)].
Huh7 and HCCLM3 cells underwent 0.25% trypsinization and were seeded on 6-well plates (1 × 105 cells per well). The oligonucleotides of miR-373-3p mimics and their nonspecific controls were bought from RiboBio (Guangzhou, China). Lentiviral vectors incorporating miR-373-3p (Lv-miR-373-3p) were synthesized in cells to represent miR-373-3p overexpression. When the cells achieved 70%–80% fusion, Lipofectamine® 3000 (Life Technologies, San Diego, CA, USA) was utilized to transfect Lv-miR-373-3p and the nonspecific control (Lv-NC), TFAP4 overexpression plasmids (TFAP4), small interfering RNA against TFAP4 (si-TFAP4), si-IGF1, and the corresponding negative control (vector, or si-NC) were transfected into the cells. All transfection steps were conducted as instructed by the supplier [19 (link)].
3
Agomir and Antagomir Transfection in T Cells
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For Agomir and Antagomir transfection, CD4+ T cells or naive CD4+ T cells were cultured under Opti-MEM (Gibco) with Agomir-21/Antagomir-21 (400 nM) or their corresponding controls for 6 hours. Then, RPMI 1640 complete medium (Gibco) was added at 200 nM to the final concentrations of Agomir-21/Antagomir-21 or their controls. For siRNA and plasmid transfection, naive CD4+ T cells were transfected with siRNA or plasmid using the Human T Cell Nucleofector Kit and Amaxa Nucleofector system (Lonza) according to the manufacturer’s protocols. Briefly, naive CD4+ T cells were collected and resuspended in 100 μL transfection reagents, and 10 μL siRNA (20 μM) or 5 μg plasmid was added and transfected into the cells by electroporation using the nucleofector program V-024 in the Amaxa Nucleofector apparatus (Lonza). After being cultured under RPMI 1640 complete medium (Gibco) for 6 hours, the cells were transferred to fresh complete medium and incubated for 48 to 72 hours and then harvested for subsequent experiments.
4
Peptide-Stimulated PBMC Expansion and IFN-γ ELISpot Assay
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PBMCs were seeded at 2 × 105 cells/well in 96-well U bottom plates in complete RPMI-1640 medium (Gibco-BRL, Waltham, MA, USA), containing either 20 ug/ml individual peptide, 20ug/ml peptide pool or no peptides (control). Every 2 days, half of the medium was replenished with complete RPMI-1640 medium (Gibco-BRL, Waltham, MA, USA), supplemented with 250 IU/ml of IL-2 (rhIL-2, #202-IL-050/CF, RnD systems, Minneapolis, MN, USA), and 50 ng/ml of IL-15 (rhIL15, #247-ILB-025/CF, RnD systems, Minneapolis, MN, USA). After 18 days, LDHC-specific T cell responses were determined by IFN-γ ELISpot assay.
5
Inhibition of Fatty Acid Synthase Modulates HIV-1 Infection in PBMCs
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PBMCs were isolated by Ficoll-Paque centrifugation, stimulated in complete RPMI-1640 medium (Gibco, Carlsbad, CA) containing 10% FBS, 100 µg/mL penicillin/streptomycin and supplemented with 5 µg/mL phytohemagglutinin (PHA; Gibco) for 48 h, and maintained thereafter in complete RPMI-1640 medium supplemented with 20 U/mL of interleukin-2 (Gibco). PBMCs were seeded in 24-well plate (2 × 105 cells/well) and triplicate wells were treated with indicated concentrations of C75, FASNALL, or with DMSO, and subsequently infected with 10 ng (p24)/mL equivalents of HIV-1NL4-3. Cells were washed 24 h post infection, and supernatants were collected and p24 content analyzed by quantitative ELISA (Zeptometrix, Buffalo, NY). PBMC viability was assessed by flow cytometry using propidium iodine (PI) exclusion (BD Pharmingen).
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PBMCs were isolated by Ficoll-Paque centrifugation, stimulated in complete RPMI-1640 medium (Gibco, Carlsbad, CA) containing 10% FBS, 100 µg/mL penicillin/streptomycin and supplemented with 5 µg/mL phytohemagglutinin (PHA; Gibco) for 48 h, and maintained thereafter in complete RPMI-1640 medium supplemented with 20 U/mL of interleukin-2 (Gibco). PBMCs were seeded in 24-well plate (2 × 105 cells/well) and triplicate wells were treated with indicated concentrations of C75, FASNALL, or with DMSO, and subsequently infected with 10 ng (p24)/mL equivalents of HIV-1NL4-3. Cells were washed 24 h post infection, and supernatants were collected and p24 content analyzed by quantitative ELISA (Zeptometrix, Buffalo, NY). PBMC viability was assessed by flow cytometry using propidium iodine (PI) exclusion (BD Pharmingen).
6
Cryopreservation and Thawing of Human PBMCs
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The total PBMCs were counted, and 3 × 106 cells were placed into each cryo-vial tube along with 0.5 ml of media A. Then, 0.5 ml of media B (20% DMSO in RPMI 1640 medium, Sigma) was added to each cryo-vial tube. The cryo-vial tubes were then sealed and placed into a cell freezing container containing isopropanol. The cells were kept at −80°C for 24 h and then put into a liquid nitrogen (LN2) canister with LN2.
When thawing frozen PBMCs, the frozen cells were quickly thawed at 37°C for 1 min. Cells were resuspended in RPMI 1640 complete medium with benzonase (25 U/ml) (Sigma). The PBMCs were then centrifuged twice at 300 g for 8 min. Finally, the cells were resuspended in 1 ml of complete RPMI 1640 medium (Gibco) without benzonase for counting, and the cell concentration was adjusted with complete RPMI 1640 medium without benzonase for the flow cytometry assay.
When thawing frozen PBMCs, the frozen cells were quickly thawed at 37°C for 1 min. Cells were resuspended in RPMI 1640 complete medium with benzonase (25 U/ml) (Sigma). The PBMCs were then centrifuged twice at 300 g for 8 min. Finally, the cells were resuspended in 1 ml of complete RPMI 1640 medium (Gibco) without benzonase for counting, and the cell concentration was adjusted with complete RPMI 1640 medium without benzonase for the flow cytometry assay.
7
Monocyte-Derived Dendritic Cell Generation
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Peripheral blood mononuclear cells (PBMCs) from a healthy donor were purchased from the American Type Culture Collection (ATCC PCS-800-011). Blood monocytes were isolated from PBMCs by plastic adhesion [17 (link)]. Mature monocyte-derived dendritic cells (moDCs) were generated in vitro from peripheral blood monocytes, according to Nair et al. [18 (link)]. Briefly, monocytes were cultured in a humidified CO2-incubator (5% CO2, 37 °C) in the complete RPMI-1640 medium (Gibco) with 10% FBS, antibiotics, and supplemented with a cytokine cocktail containing 500 U/mL rhIL-4 and 800 U/mL rhGM-CSF (both Sci-Store, Moscow, Russia). On the third day, the medium was replaced by the same fresh medium with FBS, antibiotics, and the cytokine cocktail. Induction of maturation of the immature monocyte-derived dendritic cells was performed on the 5th day by replacing the medium with the complete RPMI-1640 medium (Gibco) with 10% FBS and antibiotics, supplemented with a cytokine cocktail containing 500 U/mL rhIL-4, 800 U/mL rhGM-CSF (both Sci-Store, Moscow, Russia), and 100 U/mL rhTNF-α (Sigma-Aldrich), and subjected to subsequent incubation of the culture for 4 more days.
8
Flow Cytometry Analysis of Murine B Cell Populations
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Cells were harvested from spleen and popliteal LNs for flow cytometry and cell sorting. Splenocytes were homogenized with complete RPMI 1640 medium (Gibco), followed by red blood cell lysis in 1 ml of ACK lysing buffer (Gibco) for 1 min on ice. Popliteal LNs were homogenized with complete RPMI 1640 medium (Gibco). Cells were washed and resuspended in an appropriate volume for counting and staining. Cells were stained with the following monoclonal antibodies conjugated to fluorochromes: anti- B220, CD38, CD138, GL-7, CD21, and CD23 (eBioscience) for 30 min at 4°C in FACS staining buffer (PBS 1× with 5% FCS). Analysis and cell sort were then performed on a MoFlo (Dako-Cytomation) or Aria II (BD Biosciences). B cell populations were defined as GC (B220+ CD138− CD38lo/− GL-7+) and FO (B220+ CD138− CD21lo/− CD23+ GL-7−). Cells were collected directly in sterile tubes containing supplemented OptiMEM (Gibco) for cell culture.
9
Adoptive Transfer and Reconstitution of Immune Cells
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Mouse splenic B cells were presorted by CD19 mAb-microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). CD1dhiCD5+, CD1dintCD5+, or CD1dintCD5− (non-Breg cells) B cells were subsequently sorted by a FACSAria system (BD Biosciences) (fig. S10A). For adoptive transfer, each B cell subset (1.5 × 106 cells/0.2 ml of PBS) was transferred intravenously into recipient mice 48 hours before challenge with OXZ to induce CHS. For the reconstitution of MCs in KitW-sh/W-sh mice, bone marrow–derived MCs (BMMCs) from isolated C57BL/6 mice were cultured in complete RPMI 1640 medium containing IL-3 (10 ng/ml; PeproTech Inc., Rock Hill, NJ) for 4 weeks, following the published procedure (39 ). PeC-derived MCs (PDMCs) were cultured in complete RPMI 1640 medium containing IL-3 (10 ng/ml) and stem cell factor (30 ng/ml; PeproTech Inc., Rock Hill, NJ). After 48 hours, nonadherent cells were removed and replaced by the fresh complete medium for 9 days (40 (link)). The expression of c-Kit and FcεRI on BMMCs and PDMCs was analyzed with a FACSCanto II flow cytometer (BD Bioscience). BMMCs and PDMCs with a purity of more than 98% were used for the adoptive transfer experiments. BMMCs (1 × 107 cells per mouse, i.v., 8 weeks before challenge) or PDMCs (5 × 106 cells per mouse, i.p., 4 weeks before challenge) were injected into recipient KitW-sh/W-sh mice.
10
Spleen Lymphocyte Isolation and Proliferation Assay
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The spleens were rinsed with DPBS buffer solution containing antibiotic (1000 U/mL), which were gently triturated and filtered through a 200-mesh nylon screen [20 (link)]. The spleen cells were transferred to tubes and centrifuged at 1000 r/min for 5 min. After removed the red blood cells, the precipitation was washed repeatedly and continued to cultivate on RPMI 1640 complete medium (Thermo Fisher Scientific, Waltham, MA, USA) including 10% calf serum and 100 U/mL antibiotic in 5% CO2 incubator for 4 h at 37 °C.
The cell viability was checked using trypan blue staining (if the number above 95%, it can be used for experiments). Spleen lymphocyte suspension (2.0 × 106/mL, 100 μL) and samples (500 μg/mL, 100 μL) were added to each well in a 96-well plate, and the cells were cultured in 5% CO2 incubator at 37 °C for 36 h. MTT (5 mg/mL, 20 μL) was added and the plates were incubated for another 4 h under the same conditions. After incubation, the samples were centrifuged, and the supernatant was discard. Then DMSO (200 μL) was added to measure the OD value of each well at 570 nm.
The cell viability was checked using trypan blue staining (if the number above 95%, it can be used for experiments). Spleen lymphocyte suspension (2.0 × 106/mL, 100 μL) and samples (500 μg/mL, 100 μL) were added to each well in a 96-well plate, and the cells were cultured in 5% CO2 incubator at 37 °C for 36 h. MTT (5 mg/mL, 20 μL) was added and the plates were incubated for another 4 h under the same conditions. After incubation, the samples were centrifuged, and the supernatant was discard. Then DMSO (200 μL) was added to measure the OD value of each well at 570 nm.
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