BM cells were flushed from mouse femurs with RPMI-1640 medium containing 2% fetal bovine serum and lysed using a lysis buffer (StemCell Technologies). Then 1 × 107 of the BM cells were stained with LSK, as described previously. The cells were incubated with anti-Annexin V-PE and anti-7AAD. The cells were tested using a flow cytometer (Thermo Fisher Scientific, USA), and the flow cytometry data were analyzed using FlowJo 7.6.1 (Tree Star, Franklin Lake, NJ, USA).
Rpmi 1640 medium
Manufactured by STEMCELL
Sourced in Canada
RPMI-1640 is a widely used cell culture medium designed to support the growth of a variety of cell types, including lymphoid and myeloid cells. It provides the necessary nutrients and components for the maintenance and proliferation of cells in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (5 mentions)
Lysis buffer, by STEMCELL (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Sigenome smartpool sirna, by Horizon Discovery (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Dharmafect 1 transfection reagent, by Horizon Discovery (2 mentions)
Fetal bovine serum (fbs), by STEMCELL (2 mentions)
Cd49b positive selection kit, by STEMCELL (1 mentions)
Metformin hydrochloride, by Bio-Techne (1 mentions)
Sodium pyruvate, by STEMCELL (1 mentions)
Fura 2 am, by Merck Group (1 mentions)
P ctni, by Cell Signaling Technology (1 mentions)
Serca2, by Thermo Fisher Scientific (1 mentions)
Chir99021, by STEMCELL (1 mentions)
M csf, by STEMCELL (1 mentions)
Cd1 male mice, by Charles River Laboratories (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
M csf, by Sino Biological (1 mentions)
Streptomycin, by Sino Biological (1 mentions)
Penicillin, by Sino Biological (1 mentions)
10 protocols using rpmi 1640 medium
1
Isolation and analysis of mouse bone marrow cells
2
Culturing Lymphoma, Thymoma, and Melanoma Cells
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MHC class I-deficient lymphoma cell line RMA-S (originally generated by Klas Kärre lab, Karolinska Institute, Stockholm, Sweden and kindly provided by André Veillette, Clinical Research Institute of Montreal, Canada) and YAC-1 cells (thymoma, ATCC# TIB-160) and B16-F10 cells (melanoma, ATCC #CRL-6475) were cultured in RPMI 1640 medium (STEMCELL) containing 10% fetal bovine serum (FBS). For IL-2-activated NK cells, splenic NK cells were enriched with CD49b-positive selection kit (STEMCELL) and then were cultured in RPMI 1640 medium containing 20% FBS and 1000 IU per mL human IL-2 for 5 days.
3
Quantifying Reactive Oxygen Species in BM Cells
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BM cells were flushed from mouse femurs with RPMI-1640 medium containing 2% fetal bovine serum and lysed using a lysis buffer (StemCell Technologies). Then, 1 × 107 of the BM cells were stained with LSK, as described previously. Next, the cells were incubated with DCF (10 μM) for 30 min at 37 °C. After washing with PBS, the cells were analyzed using a flow cytometer (Thermo Fisher Scientific, USA).
4
NSCLC Cell Culture and Manipulation
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A549 and H1651 human non-small cell lung cancer (NSCLC) cells were purchased from American Type Culture Collection (Manassas, VA, USA). Cells were preserved in liquid nitrogen after shipment and were used on passage 2 to 5. A549 and H1651 cells were cultured in RPMI-1640 medium (STEMCELL Technologies, Vancouver, Canada) supplemented with 10% FBS (STEMCELL Technologies) and 100 U/mL of penicillin/streptomycin (Thermo Fisher Scientific, Waltham, MA, USA) in a cell incubator with 37°C, 5% CO2 atmosphere. α4 overexpression in A549 or H1651 cells was achieve by lentiviral transfection, and A549 or H1651 cell line with stable knockdown of A/B catalytic subunit of PP2A was constructed by lentiviral transfection of targeting shRNA. Lentiviral vectors described above were constructed by Genecopoeia (Rockville, MD, USA) and were used following manufacturer’s instructions. Metformin hydrochloride (Tocris Bioscience, Bristol, UK) was pre-diluted in complete culture medium as 10× stock and preserved under −8°C before use. OA (Tocris Bioscience) was pre-diluted in DMSO as 100× stock and preserved under −20°C before use.
5
Cardiomyocyte Differentiation and Analysis
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High-glucose Dulbecco’s Modified Eagle Medium (DMEM) and RPMI 1640 Medium (ATCC modification), B-27 supplements with and without insulin, TrypLE, Dulbecco’s PBS (DPBS), fetal bovine serum (FBS), penicillin-streptomycin (P/S), non-essential amino acids (NEAA), G-glutamine, and sodium pyruvate were purchased from ThermoFisher Scientific. CHIR99021, IWP4, and mTeSR were from STEMCELL Technologies. FURA 2/AM and ISO were from Sigma-Aldrich. Primary antibodies included SERCA2 (Invitrogen MA3-910), cTnI (Proteintech 66376-1-IG), p-cTnI (Cell Signaling Technology 4004S), p-PLN (Invitrogen PA5-38317), PLN (Invitrogen MA3-922), and TPM11 (Invitrogen PA5-29846). Secondary antibodies were from Bio-Rad.
6
Murine Bone Marrow-Derived Macrophage Generation
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BMDMs were generated by collecting femur and tibia from the hind legs of 6- to 8-week-old CD1 male mice (Charles River). Muscles attached to bones were removed using sterile scissors and forceps. Bone marrow was isolated by flushing the marrow into a sterile 50-mL tube with a syringe filled with RPMI 1640 medium. Upon centrifugation at 2,000 rpm for 5 min, cells were resuspended in RPMI 1640 medium supplemented with 10% FBS, macrophage colony-stimulating factor (M-CSF; 20 ng/mL; StemCell Technologies), 2% penicillin/streptomycin, and 2 mM l -glutamine (Gibco). Subsequently, the cells were plated on 90-mm nontissue culture-treated plates at a density of 5 × 106 cells/mL and incubated at 37°C in a 5% CO2 atmosphere. Three days after cell seeding, an extra 10 mL of fresh RPMI 1640 (supplemented with 10% FBS, M-CSF [20 ng/mL], 2% penicillin/streptomycin, and 2 mM l -glutamine) was added to each plate, and incubation continued for an additional 3 days. On the sixth day, supernatants were discarded. The attached cells were resuspended in RPMI 1640 after being dislodged by trypsin-EDTA. Finally, the cells were counted, and approximately 1.5 × 106 cells were seeded in tissue culture plates for 24 h before the experimental procedure. The culture medium was replaced by RPMI 1640 growth medium with 10% FBS.
7
Isolation and Culture of Human and Mouse Macrophages
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Peripheral blood mononuclear cells (PBMCs) were isolated from human blood using Ficoll-Paque (GE Healthcare, Chicago, IL, USA). Human monocyte-derived macrophages were purified by positive selection of CD14 and CD16 cells from the PBMCs using MACS Microbeads from Miltenyi Biotec (Leiden, Netherlands), following the manufacturer's protocol. The isolated monocytes were then induced with 20 ng/mL of macrophage colony-stimulating factor (R&D Systems, Minneapolis, USA) in RPMI-1640 medium supplemented with 10% fetal bovine serum (STEMCELL Technologies), 100 U/mL of penicillin/streptomycin (Thermo Fisher Scientific), 10 mM Glutamax, and 10 mM pyruvate. Mouse alveolar macrophages (AMs) and bone marrow-derived macrophages (BMDMs) were isolated from control mice as previously describe [39 (link), 40 (link)] and cultured in RPMI-1640 medium supplemented with 10% (v/v) fetal calf serum and 2 mM glutamine with penicillin (100 U/mL)/streptomycin (100 mg/mL) and M-CSF (10 ng/mL, Sino Biological, 51112-M08H). The cells were grown in 96-well plates (200 μL final volume; Corning Inc., Corning, NY, USA) and incubated at 37 °C in a humidified incubator with 5% CO2.
8
Synergistic Potential of Cord Blood and Placental Cells
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Example 17
Total nucleated cells are isolated from a unit of cord blood by Hetastarch separation. Total nucleated placental cells are obtained from 750 milliliters of placental perfusate by Ficoll separation. The total nucleated cells from placenta and cord blood are combined in triplicate in 35 mm culture dishes in Methocult GF+ H4435 medium (Stem Cell Technologies, Vancouver, Canada), or RPMI 1640 medium supplemented with 2% fetal calf serum and 1% Stemspan CC100 cytokine cocktail (Stem Cell Technologies, Vancouver, Canada). Cells are combined in at least two ratios (e.g., 2×105:2×105; 1×105:3×105; 3×105:1×105), and are cultured for 14 days. The morphology of the cells is then examined under phase contrast microscope, and the total number of colony-forming units (e.g., CFU-GM, CFU-L, CFU-M, CFU-G, CFU-DC, CFU-GEMM, CFU-E) are recorded. A determination is then made as to which ratio produces the highest number of colony-forming units.
9
ARID1A Knockout in RMG1 Cells
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RMG1 cells were cultivated in RPMI-1640 medium (Stemcell technologies) supplemented with 10% fetal bovine serum (Life Technologies) in 5% CO2 at 37°C. ARID1A was knocked out in RMG1 cells using CRISPR/cas9 technology with gRNA targeting exon 2 of ARID1A gene (5’-CTTGCTGCGGTCCTGACGGAGG-3’). Targeted sequencing with Illumina MiSeq confirmed homozygous deletion (c.1615 del C) in RMG1_AC14 ARID1A knockout single clone. For RNA interference, cells were transfected with siGENOME-SMARTpool siRNAs from Dharmacon (Non-targeting siRNA Pool #1 as si-Cont and si-ARID1A, catalog #D-001206-13 and #M-017263-01). Transfections were done with Dharmafect1 transfection reagent (Dharmacon) according to manufacturer’s protocol and harvested 48 hours after the siRNA administration. For experiments with overexpression of GFP or nuclear-targeting GFP-RNaseH1 (gift from R. Crouch), transfections were performed with Lipofectamine 3000 (Invitrogen) according to manufacturer’s instructions 24 hours after the siRNA transfections. Detailed catalog number and dilution information for all antibodies used in the manuscript can be found in S2 Table
10
ARID1A Knockout in RMG1 Cells
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Cell culture and transfection RMG1 cells were cultivated in RPMI-1640 medium (Stemcell technologies) supplemented with 10% fetal bovine serum (Life Technologies) in 5% CO 2 at 37°C. ARID1A was knocked out in RMG1 cells using CRISPR/cas9 technology with gRNA targeting exon 2 of ARID1A gene (5'-CTTGCTGCGGTCCTGACGGAGG-3'). Targeted sequencing with Illumina MiSeq confirmed homozygous deletion (c.1615 del C) in RMG1_AC14 ARID1A knockout single clone.
For RNA interference, cells were transfected with siGENOME-SMARTpool siRNAs from Dharmacon (Non-targeting siRNA Pool #1 as si-Cont and si-ARID1A). Transfections were done with Dharmafect1 transfection reagent (Dharmacon) according to manufacturer's protocol and harvested 48 hours after the siRNA administration. For experiments with overexpression of GFP or nuclear-targeting GFP-RNaseH1 (gift from R. Crouch), transfections were performed with Lipofectamine 3000 (Invitrogen) according to manufacturer's instructions 24 hours after the siRNA transfections.
For RNA interference, cells were transfected with siGENOME-SMARTpool siRNAs from Dharmacon (Non-targeting siRNA Pool #1 as si-Cont and si-ARID1A). Transfections were done with Dharmafect1 transfection reagent (Dharmacon) according to manufacturer's protocol and harvested 48 hours after the siRNA administration. For experiments with overexpression of GFP or nuclear-targeting GFP-RNaseH1 (gift from R. Crouch), transfections were performed with Lipofectamine 3000 (Invitrogen) according to manufacturer's instructions 24 hours after the siRNA transfections.
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