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MB Sample ID: SA323544
Local Sample ID: | R_B24h_EA_21_1_2 |
Subject ID: | SU003095 |
Subject Type: | Cultured cells |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Genotype Strain: | MDA-MB-231 cells |
Gender: | Not applicable |
Cell Biosource Or Supplier: | ATCC (Manassas, VA, USA); ATCC HTB-26 |
Cell Strain Details: | Epithelial breast cancer cells; absence of estrogen and progesterone receptors, HER2 overexpression |
Cell Passage Number: | Inferior to 10 |
Cell Counts: | 5 M |
Select appropriate tab below to view additional metadata details:
Subject:
Subject ID: | SU003095 |
Subject Type: | Cultured cells |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Genotype Strain: | MDA-MB-231 cells |
Gender: | Not applicable |
Cell Biosource Or Supplier: | ATCC (Manassas, VA, USA); ATCC HTB-26 |
Cell Strain Details: | Epithelial breast cancer cells; absence of estrogen and progesterone receptors, HER2 overexpression |
Cell Passage Number: | Inferior to 10 |
Cell Counts: | 5 M |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
R_B24h_EA_21_1_2 | SA323544 | FL038653 | Resistant_Pd2Spm_treated_24h | Treatment_group |
Collection:
Collection ID: | CO003088 |
Collection Summary: | MDA-MB-231 parental and MDA-MB-231/R cDDP-resistant cell lines were seeded at a density of 3 × 10^4 cells/cm2 onto 13.55 cm diameter Petri dishes, cultured in a humidified atmosphere of 5% CO2 at 37 ◦C and allowed to adhere for 24 h (t = 0 h). After this, the experiment was initiated by adding stock solutions of each drug to achieve the corresponding half of maximal inhibitory effect IC50 values: 1.0 µM cDDP and 7.9 μM Pd2Spm. Phosphate-buffer saline was used in control groups. Then, cells were incubated and collected at t = 24 h and t = 48 h, with basis on the population (25.5 ± 0.9 h and 30.6 ± 1.1 h for MDA-MB-231 and MDA-MB-231/R cells, respectively). At each time-point, cells were harvested using a 0.25% (v/v) trypsin-EDTA solution, washed twice with PBS and centrifuged (300 g, 5 min, 20 ◦C). The cell pellet was directly stored at − 80 ◦C until analysis. Three independent experiments with triplicates were performed for each cell type and time-point. |
Sample Type: | Epithelial cells |
Collection Method: | Trypsinization for cells harvesting followed by pellet collection and storage at − 80 ◦C (until extract prepatation) |
Collection Frequency: | One collection per sample replicate |
Collection Duration: | Between 2 and 5 minutes |
Storage Conditions: | -80℃ |
Treatment:
Treatment ID: | TR003104 |
Treatment Summary: | Cells were treated during culture time by adding drugs stock solution to the growth culture medium. Briefly, after seeding, cells were allowed to adhere for 24 h (t = 0 h). After this, the experiment was initiated by adding stock solutions of each drug to achieve the corresponding half of maximal inhibitory effect IC50 values: 1.0 µM cDDP and 7.9 μM Pd2Spm. Phosphate-buffered saline (PBS) was used in control groups. Then, cells were incubated and collected at t = 24 h and t = 48 h, with basis on the population (25.5 ± 0.9 h and 30.6 ± 1.1 h for MDA-MB-231 and MDA-MB-231/R cells, respectively). |
Treatment Compound: | cDDP or Pd2Spm (PBS for controls) |
Treatment Route: | Dissolution of drugs stock solutions into the culture medium |
Treatment Dose: | 1.0 µM cDDP and 7.9 μM Pd2Spm |
Treatment Dosevolume: | 5.5 mL |
Treatment Doseduration: | 24 h and 48 h |
Treatment Vehicle: | Phosphate-buffered saline (PBS) |
Cell Media: | Dulbecco’s Modified Eagle’s Medium—high-glucose cell growth medium (DMEM-HG) |
Cell Envir Cond: | Humidified atmosphere of 5% CO2 at 37 ◦C |
Cell Harvesting: | Tripsinization |
Cell Pct Confluence: | ~ 70 to 80% |
Sample Preparation:
Sampleprep ID: | SP003101 |
Sampleprep Summary: | The cellular polar extracts were extracted using a biphasic extraction method of methanol/chloroform/water. Basically, cell pellets were resuspended in 650 µL of 80% (v/v) methanol-miliQ water solution, transferred to microcentrifuge tubes with 150 mg of glass beads, and vortexed for 5 min. Subsequently, 260 µL of 100% chloroform and 260 µL of 100% chloroform plus 220 µL MiliQ water were added to samples, which were vortexed for 5 min between solvents addition. The samples were kept at − 20 °C for 10 min and centrifuged. The aqueous phase of the resulting extract was collected into a new tube, vacuum-dried and stored at − 80 °C until the NMR analysis. All samples and reagents were kept in ice during the extraction procedure. Before NMR analysis, the dry aqueous extracts were suspended in 650 µL of 100 mM sodium phosphate buffer (pH 7.4, in D2O containing 0.25% 3-(trimethylsilyl)propionic-2,2,3,3-D4 acid sodium salt (TSP-D4) for chemical shift referencing) and transferred into 5 mm NMR tubes. |
Processing Storage Conditions: | On ice |
Extraction Method: | Biphasic extraction method of methanol/chloroform/water |
Extract Storage: | -80℃ |
Sample Resuspension: | Dry aqueous extracts were suspended in 650 µL of 100 mM sodium phosphate buffer (pH 7.4, in D2O containing 0.25% 3-(trimethylsilyl)propionic-2,2,3,3-D4 acid sodium salt (TSP-D4) for chemical shift referencing) |
Analysis:
MB Sample ID: | SA323544 |
Analysis ID: | AN004901 |
Analysis Type: | NMR |
Acquisition Date: | March 2023 |
Software Version: | Topspin 3.6.5 |
Operator Name: | Tatiana João Carneiro |
Results File: | ST002982_AN004901_Results.txt |
Units: | ppm |
NMR:
NMR ID: | NM000270 |
Analysis ID: | AN004901 |
Instrument Name: | Avance III HD |
Instrument Type: | FT-NMR |
NMR Experiment Type: | 1D-1H |
NMR Comments: | 1st subfolder contains raw spectra; 2nd subfolder contains manually processed spectra |
Field Frequency Lock: | D2O |
Spectrometer Frequency: | 500 MHz |
NMR Probe: | 5 mm TXI probe |
NMR Solvent: | 100% D2O |
NMR Tube Size: | 5mm |
Shimming Method: | Automatic and manual |
Receiver Gain: | 203 |
Chemical Shift Ref Cpd: | TSP-D4 |
Temperature: | 298K |
Number Of Scans: | 512 |
Dummy Scans: | 4 s |
Acquisition Time: | 2.34 s |
Relaxation Delay: | 2 s |
Spectral Width: | 7002.801 Hz |
Num Data Points Acquired: | 32 k |
Real Data Points: | 64 k |
Line Broadening: | 0.3 Hz (multiplication) |
Baseline Correction Method: | Manual |
Chemical Shift Ref Std: | TSP-D4 |
NMR Results File: | TNBC_cells_untreated_treated_EA_NMR.txt UNITS:ppm |