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MB Sample ID: SA308592
Local Sample ID: | Pos_KO_4 |
Subject ID: | SU002962 |
Subject Type: | Cultured cells |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Species Group: | Mammals |
Select appropriate tab below to view additional metadata details:
Subject:
Subject ID: | SU002962 |
Subject Type: | Cultured cells |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Species Group: | Mammals |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
Pos_KO_4 | SA308592 | FL037159 | Knockout | Genotype |
Collection:
Collection ID: | CO002955 |
Collection Summary: | Primary mus musculus cells |
Sample Type: | Osteoclast |
Treatment:
Treatment ID: | TR002971 |
Treatment Summary: | All in vitro experiments were performed at least 3 times. Primary bone marrow macrophages (BMMs) were prepared as described. Marrow was extracted from femora and tibiae of 6- to 8-week-old mice with α minimum essential medium (α-MEM) and cultured in α-MEM containing 10% inactivated fetal bovine serum, 100 IU/mL penicillin, and 100 μg/mL streptomycin (α-10 medium) with 1:10 of mMCSF producing cell line, CMG 14-12 condition media on petri-plastic dishes. Cells were incubated at 37°C in 5% CO2 for 3 days and then washed with phosphate-buffered saline (PBS) and lifted with 1X trypsin/EDTA in PBS. A total of 1.2 × 104 BMMs were cultured in 500 μL α-MEM containing 10% heat-inactivated fetal bovine serum with glutathione-S transferase–RANKL and 30 ng/mL of mouse recombinant macrophage colony-stimulating factor (M-CSF) in 48-well tissue culture plates, some containing sterile bovine bone slices. |
Sample Preparation:
Sampleprep ID: | SP002968 |
Sampleprep Summary: | Cells were quenched with cold LC/MS-grade methanol, then scraped and transferred to Eppendorf tubes. Samples were dried in a SpeedVac. The samples were then reconstituted in 1 mL of cold methanol:acetonitrile:water (2:2:1) and subjected to three cycles of vortexing, freezing in liquid nitrogen, and 10 min of sonication at 25 °C. Samples were stored at −20 °C overnight and then centrifuged for 10 min at 14,000×g and 4 °C. Supernatants were transferred to new tubes and dried by a SpeedVac. The protein abundance of each sample was determined by using BCA. A quantity of 1 μl of acetonitrile:water (2:1) per each 2.5 μg of protein was used. Samples were subjected to two cycles of vortexing and 10 min of sonication at 25 °C. Next, samples were centrifuged for 10 min at 14,000×g and 4 °C, transferred supernatant to LC vials, and stored at −80 °C until MS analysis |
Combined analysis:
Analysis ID | AN004668 | AN004669 |
---|---|---|
Analysis type | MS | MS |
Chromatography type | HILIC | HILIC |
Chromatography system | Thermo Vanquish Flex UHPLC Systems | Thermo Vanquish Flex UHPLC Systems |
Column | HILICON iHILIC-(P) Classic (100 x 2.1mm,5um) | HILICON iHILIC-(P) Classic (100 x 2.1mm,5um) |
MS Type | ESI | ESI |
MS instrument type | Orbitrap | Orbitrap |
MS instrument name | Thermo Orbitrap ID-X Tribrid | Thermo Orbitrap ID-X Tribrid |
Ion Mode | NEGATIVE | POSITIVE |
Units | peak area | peak area |
Chromatography:
Chromatography ID: | CH003514 |
Instrument Name: | Thermo Vanquish Flex UHPLC Systems |
Column Name: | HILICON iHILIC-(P) Classic (100 x 2.1mm,5um) |
Column Temperature: | 45 |
Flow Gradient: | 0–1 min: 90% B, 1–12 min: 90-35% B, 12–12.5 min: 35-25% B, 12.5–14.5 min: 25% B |
Flow Rate: | 250 uL/min |
Solvent A: | 20 mM ammonium bicarbonate, 0.1% ammonium hydroxideand 2.5 μM medronic acid in water:acetonitrile (95:5) |
Solvent B: | acetonitrile:water (95:5) |
Chromatography Type: | HILIC |
MS:
MS ID: | MS004415 |
Analysis ID: | AN004668 |
Instrument Name: | Thermo Orbitrap ID-X Tribrid |
Instrument Type: | Orbitrap |
MS Type: | ESI |
MS Comments: | Data were collected with the following MS source settings: spray voltage, -2.8 kV; sheath gas, 50; auxiliary gas, 10; sweep gas, 1; ion transfer tube temperature, 300°C; vaporizer temperature, 200°C; mass range, 67 – 1000 Da; resolution, 120,000; maximum injection time, 200 ms; isolation window, 1.5 Da. XCMS and Skyline software were used for data processing |
Ion Mode: | NEGATIVE |
MS ID: | MS004416 |
Analysis ID: | AN004669 |
Instrument Name: | Thermo Orbitrap ID-X Tribrid |
Instrument Type: | Orbitrap |
MS Type: | ESI |
MS Comments: | Data were collected with the following MS source settings: spray voltage, 3.5 kV; sheath gas, 50; auxiliary gas, 10; sweep gas, 1; ion transfer tube temperature, 300°C; vaporizer temperature, 200°C; mass range, 67 – 1000 Da; resolution, 120,000; maximum injection time, 200 ms; isolation window, 1.5 Da. XCMS and Skyline software were used for data processing |
Ion Mode: | POSITIVE |