To obtain murine bone marrow-derived macrophages (BMDMs), marrow was flushed from the femurs and tibia of mice, and the hematopoietic stem cells were allowed to differentiate for 7 days in Dulbecco’s modified Eagle medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco), 1% penicillin-streptomycin (Pen-Strep) solution (Gibco), and 20% L929 conditioned medium. After 7 days, the BMDMs were harvested using Ca2+/Mg2+-free phosphate-buffered saline (PBS) (Gibco) containing 5 mM EDTA (Invitrogen, Life Technologies), and maintained in DMEM containing 10% FBS and 10% L929 conditioned medium after infection. Immortalized BMDMs (iBMDMs) were immortalized by infection with the J2 retrovirus (BEI Resources). RAW 264.7 and THP-1 cells were obtained from American Type Tissue Collection (ATCC) and were maintained in DMEM and RPMI 1640, respectively, with 10% FBS. THP-1 differentiation was induced using 20 ng/ml phorbol 12-myristate 13-acetate (PMA) (Sigma) for 18 to 20 h.
Ca2 mg2 free phosphate buffered saline
Manufactured by Thermo Fisher Scientific
Ca2+/Mg2+-free phosphate buffered saline (PBS) is an isotonic buffer solution used to maintain the pH and osmolarity of cell cultures and other biological samples. It serves as a basic medium for dilution, washing, and suspending cells without affecting their viability or function.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Glutamax, by Thermo Fisher Scientific (4 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Dmem low glucose medium, by Thermo Fisher Scientific (2 mentions)
Non essential amino acid solution, by Merck Group (2 mentions)
Pen strep solution, by Thermo Fisher Scientific (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by American Type Culture Collection (1 mentions)
Vitronectin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Complete e8 medium, by Thermo Fisher Scientific (1 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Lonza (1 mentions)
Penicilin, by Thermo Fisher Scientific (1 mentions)
75 cm2 flask, by BD (1 mentions)
Glutamine, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
5 protocols using ca2 mg2 free phosphate buffered saline
1
Murine Bone Marrow-derived Macrophage Isolation
2
Isolating and Culturing Chicken Embryonic Stem Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The area pellucida was isolated from Stage X (EG&K) embryos of Hyline chicken (Gallus gallus) as previously described [33 ], washed twice with DMEM/F-12 medium (Gibco, Carlsbad, CA) and dispersed gently using a 1000-μl pipette into a single cell suspension. The cells were centrifuged at 350× g for 5 min, the supernatant was removed, and cells were resuspended in complete E8 medium (Gibco) containing 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco). The cells were seeded at a density of 2 × 104 cells/cm2 in Vitronectin (Gibco)-coated culture plates (Costar, Corning, NY). When cells grew to 70–80% confluence, passaging was performed by washing and pipetting the cells with Ca2+/Mg2+-free phosphate buffered saline (PBS; Gibco).
3
Isolation and Culture of Human Bone Marrow-Derived Mesenchymal Stem Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bone marrow aspirates were obtained by percutaneous direct aspiration from the iliac crest of 5 healthy volunteers at University Hospital Virgen de la Arrixaca (Murcia, Spain). Bone marrow was collected with 20 U/ml sodium heparin, followed by a Ficoll density gradient-based separation by centrifugation at 540 g for 20 min. After, mononuclear cell fraction was collected, washed twice with Ca2+/Mg2+-free phosphate buffered saline (PBS) (Gibco Invitrogen) and seeded into 175-cm2 culture flasks (Nunc, Thermo Fisher Scientific) at a cell density 1.5 × 105 cells/cm2 in serum-containing media, composed of DMEM low glucose medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Lonza), 1% GlutaMAX (Thermo Fisher Scientific), non-essential amino acid solution (Sigma-Aldrich) and 1% penicillin/streptomycin (Thermo Fisher Scientific). After 3 days of culture at 37 °C and 7% CO2, non-attached cells were removed and fresh complete medium was added. Culture media were renewed every 2 days, and the isolated hBMSCs were passaged when cultures were 70–80% confluent. All studies were performed using hBMSCs expanded within culture passages 3–4.
4
Pancreatic Islet Cell Isolation and Culturing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The pancreatic islets were isolated as described previously [26 (link)] and cultured in RPMI 1640 (Invitrogen/GIBCO, Grand Island, NY, USA) with glucose 2 g/L supplemented with 10% fetal bovine serum (FBS; Invitrogen/GIBCO), 100 IU/mL penicilin-100 μg/mL streptomycin (Invitrogen/GIBCO) and glutamine (2 mmol/L; Invitrogen/GIBCO) at 37℃ in a humidified air atmosphere containing 5% CO2. Some islets immediately adhered to the surfaces of the flasks. Within several days, a monolayer of cells was observed growing out and away from the islets and after 13 to 15 days of culturing, cells in the monolayer reached to 70% confluency and named as passage zero (P0) cells. For passaging, the cells were washed with Ca2+-Mg2+ free phosphate-buffered saline (PBS) (Invitrogen/GIBCO) and detached by incubating with 0.25% trypsin-ethylenediaminetetraacetic acid solution (Invitrogen/GIBCO) for 5–10 minutes at 37℃. After addition of growth medium to inactivate trypsin, the cells were then centrifugated at 200 g for 10 minutes, resuspended in 1 mL complete medium, counted in duplicate using Thoma chamber and then plated in 75 cm2 flasks (BD Biosciences, San Diego, CA, USA) at densities of 1×106 cells/flask. The growth medium was replaced every 3 days over a 10–14 day period.
5
Isolation and Expansion of hMB-MSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A standard protocol for the isolation and expansion of hMB-MSCs was used as previously described [40 (link)]. Bone marrow aspirates were obtained by percutaneous direct aspiration from the iliac crest of 5 healthy volunteers at University Hospital Virgen de la Arrixaca (Murcia, Spain). Bone marrow was collected with 20 U/ml sodium heparin, followed by Ficoll density gradient-based separation by centrifugation at 540 g for 20 min. After, the mononuclear cell fraction was collected, washed twice with Ca2+/Mg2+-free phosphate buffered saline (PBS) (Gibco Invitrogen) and seeded into 175-cm2 culture flasks (Nunc, Thermo Fisher Scientific) at a cell density of 1.5 × 105 cells/cm2 in serum-containing media (designated as the basal media), composed of DMEM low glucose medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Lonza), 1% GlutaMAX (Thermo Fisher Scientific), nonessential amino acid solution (Sigma‒Aldrich) and 1% penicillin/streptomycin (Thermo Fisher Scientific). After 3 days of culture at 37 °C and 7% CO2, nonadherent cells were removed, and fresh complete medium was added. Culture media were renewed every 2 days, and the isolated hMB-MSCs were passaged when cultures were 70–80% confluent. All studies were performed using hMB-MSCs expanded within culture passages 3–4.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!