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MB Sample ID: SA323588

Local Sample ID:S_C24h_EA_13_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

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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 IDMB Sample IDFactor Level IDLevel ValueFactor Name
S_C24h_EA_13_1_2SA323588FL038658Sensitive_Controls_24hTreatment_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:SA323588
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
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