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MB Sample ID: SA154559
Local Sample ID: | KD-D2 |
Subject ID: | SU001755 |
Subject Type: | Cultured cells |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Gender: | Not applicable |
Select appropriate tab below to view additional metadata details:
Subject:
Subject ID: | SU001755 |
Subject Type: | Cultured cells |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Gender: | Not applicable |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
KD-D2 | SA154559 | FL018221 | UGCG-knockdown | Treatment |
KD-D2 | SA154559 | FL018221 | Test | Types |
Collection:
Collection ID: | CO001748 |
Collection Summary: | CD4+ T-cells were isolated from human umbilical cord blood as described previously (Khan et al., 2020; Tripathi et al., 2017; Ubaid et al., 2018). CD4+ T-cells were isolated from human umbilical cord blood as described previously [1-3]. 1. Ubaid, U. et al. Transcriptional Repressor HIC1 Contributes to Suppressive Function of Human Induced Regulatory T Cells. Cell Rep 22, 2094-2106, doi:10.1016/j.celrep.2018.01.070 (2018). 2. Khan, M. M. et al. CIP2A Constrains Th17 Differentiation by Modulating STAT3 Signaling. iScience 23, 100947, doi:10.1016/j.isci.2020.100947 (2020). 3. Tripathi, S. K. et al. Genome-wide Analysis of STAT3-Mediated Transcription during Early Human Th17 Cell Differentiation. Cell Rep 19, 1888-1901, doi:10.1016/j.celrep.2017.05.013 (2017). |
Sample Type: | T-cells |
Storage Conditions: | -80℃ |
Treatment:
Treatment ID: | TR001768 |
Treatment Summary: | For Th17 cell differentiation, isolated CD4+ cells were activated with a combination of plate-bound anti-CD3 (750 ng/24-well culture plate well; Immunotech/Beckman Coulter REF # IM-1304) and soluble anti-CD28 ((1ug/mL; Immunotech/Beckman coulter REF # IM1376) antibodies in serum-free X-Vivo 20 medium (Lonza), in the absence (Th0) or presence (Th17) of IL-6 (20ng/ml, Roche, Cat# 11138600 001); IL-1β (10ng/ml, R&D Systems Cat # 201 LB); TGF-β1 (10ng/ml, R&D Systems Cat# 240); anti-IL-4 (1 g/ml) R&D Systems Cat# MAB204) and anti-IFN-γ (1 μg/ml R&D Systems Cat#MAB-285). Differentiation of Th17 cells was confirmed by measuring IL-17 expression by quantitative real-time PCR, at 72 hours of Th17 / Th0 culturing. For iTreg cell culturing, after of CD25+ cells, done using LD columns and a CD25 depletion kit (Miltenyi Biotec), CD4+CD25− cells were activated with plate-bound anti-CD3 (500 ng/24-well culture plate well) and soluble anti-CD28 (500 ng/mL) at a density of 2 × 106 cells/mL of X-vivo 15 serum-free medium (Lonza). For iTreg differentiation, the medium was supplemented with IL-2 (12 ng/mL), TGF-β (10 ng/mL) (both from R&D Systems), all-trans retinoic acid (ATRA) (10 nM; Sigma-Aldrich), and human serum (10%) and cultured at 37°C in 5% CO2. Control Th0 cells were stimulated with plate-bound anti-CD3 soluble anti-CD28 antibodies without cytokines. For confirmation of iTreg cell differentiation, we used intracellular staining to measure, at 72 hours of iTreg culturing, expression of FOXP3 which is the major transcription factor driving Treg differentiation. Intracellular staining was performed using buffer sets of Human Regulatory T-cell Staining Kit (eBioscience/Thermo Fisher Scientific), following the manufacturer’s protocol. The following antibodies were used: anti-human FOXP3-PE (eBioscience, Cat. No. 12-4776-42) and rat IgG2a isotype control (eBioscience, Cat. No. 72-4321-77A). All samples were acquired by a flow cytometer (LSRII) and analyzed either with FlowJo (FLOWJO, LLC) or with Flowing Software. For Th1 and Th2 cells, purified naive CD4+ T-cells were activated with plate-bound anti-CD3 (500 ng/24-well culture plate well) and 500 ng/ml soluble anti-CD28 and cultured in the absence (Th0) or presence of 2.5 ng/ml IL-12 (R&D Systems) (Th1) or 10 ng/ml IL-4 (R&D Systems) (for Th2). At 48 hours following the activation of the cells, 17 ng/ml IL-2 (R&D Systems) was added to the cultures. Differentiation of Th1 and Th2 cells was confirmed by measuring (using flow cytometry) the expression of T-bet and Gata3 at 72 hours after cell activation. Briefly, cells were fixed and permeabilized using the Intracellular Fixation & Permeabilization Buffer Set (eBioscience / Thermo Fisher Scientific), according the manufacturer’s protocol. The following antibodies were used: anti-human GATA3-PE (eBioscience, 12-9966), anti-human T-bet-BV711 (BD, 563320) and corresponding isotype controls (BV711 Mouse IgG1, BD, 563044 and PE Rat IgG2b, eBioscience, 12-4031-82). Samples were acquired by BD LSRFortessa™ cell analyzer and data were analyzed using FlowJo software (FLOWJO, LLC). |
Sample Preparation:
Sampleprep ID: | SP001761 |
Sampleprep Summary: | The frozen cell preps were defrosted on ice. The samples were extracted using a modified Folch method[1]. 1. Sen, P. et al. Persistent Alterations in Plasma Lipid Profiles Before Introduction of Gluten in the Diet Associated With Progression to Celiac Disease. Clin Transl Gastroenterol 10, 1-10, doi:10.14309/ctg.0000000000000044 (2019). Briefly, 120 µL of cold (4 °C) extraction solvent (CHCl3: MeOH, (2:1 v/v) was added to the samples. The extraction solvent containing the following internal standards: C17 Lactosyl(beta) ceramide (D18:1/17:0, 20 ppb), C17 Glucosyl(beta) ceramide (D18:1/17:0, 20 ppb), C17 ceramide (D18:1/17:0, 20 ppb), C16 ceramide-d7 (d18:1-d7/16:0, 16,57 ppb), C18 ceramide-d7 (d18:1-d7/18:0, 8.75 ppb), C24 ceramide-d7 (d18:1-d7/24:0, 20 ppb), and C24:1 ceramide-d7 (d18:1-d7/24:1(15Z), 9,96 ppb). The samples were the vortexed briefly and left on ice for 30 minutes. The samples were then centrifuged (9400g, 5 min, 4 °C) and then 60 µL of the bottom layer was transfer to a clean mass spectrometry vial (2 mL). The samples were then stored at –80 °C. |
Combined analysis:
Analysis ID | AN002736 |
---|---|
Analysis type | MS |
Chromatography type | Reversed phase |
Chromatography system | ACQUITY UHPLC |
Column | Waters BEH C18 (100 × 2.1 mm,1.7um) |
MS Type | ESI |
MS instrument type | Triple quadrupole |
MS instrument name | ABI Sciex 5500 QTrap |
Ion Mode | POSITIVE |
Units | ng/ml |
Chromatography:
Chromatography ID: | CH002022 |
Chromatography Summary: | Chromatographic separation was performed on an ACQUITY UHPLC BEH C18 column (2.1 mm × 100 mm, particle size 1.7 µm, Waters, Milford, MA, USA). The flow rate was set at 0.4 ml/min throughout the run with an injection volume of 1 µL. The following solvents were used for the gradient elution: Solvent A was H2O with 1% NH4Ac (1M) and HCOOH (0.1%) added. Solvent B was a mixture of ACN: IPA (1:1 v/v) with 1% NH4Ac (1M) and HCOOH (0.1%) added. The gradient was programmed as follows: 0 to 2 min 35-80% B, 2 to 7 min 80-100 % B, 7 to 14 min 100% B. The column was equilibrated with a 7min period of 35 % B prior to the next run. |
Instrument Name: | ACQUITY UHPLC |
Column Name: | Waters BEH C18 (100 × 2.1 mm,1.7um) |
Flow Gradient: | The gradient was programmed as follows: 0 to 2 min 35-80% B, 2 to 7 min 80-100 % B, 7 to 14 min 100% B. The column was equilibrated with a 7min period of 35 % B prior to the next run. |
Flow Rate: | 0.4ml/min |
Solvent A: | 100% water; 0.1% formic acid; 1% ammonium acetate |
Solvent B: | 50% acetonitrile/50% isopropanol; 0.1% formic acid; 1% ammonium acetate |
Chromatography Type: | Reversed phase |
MS:
MS ID: | MS002533 |
Analysis ID: | AN002736 |
Instrument Name: | ABI Sciex 5500 QTrap |
Instrument Type: | Triple quadrupole |
MS Type: | ESI |
MS Comments: | The ceramides were quantified using a targeted multiple reaction monitoring (MRM) method using UHPLC as a separation technique. The LC separation was based on the global lipidomics method previously described 71. Briefly, the UHPLC was a Exion AD (Sciex) integrated system. The samples were held in a cool box at 15 °C prior to the analysis. The needle was washed with both a 10% DCM in MeOH and ACN: MeOH: IPA: H2O (1:1:1:1 v/v/v/v) with 1% HCOOH for a total of 7.5 seconds each. The solvents were delivered using a quaternary solvent and a column oven (set to 50 °C). The separation was performed on an ACQUITY UHPLC BEH C18 column (2.1 mm × 100 mm, particle size 1.7 µm, Waters, Milford, MA, USA). The flow rate was set at 0.4 ml/min throughout the run with an injection volume of 1 µL. The following solvents were used for the gradient elution: Solvent A was H2O with 1% NH4Ac (1M) and HCOOH (0.1%) added. Solvent B was a mixture of ACN: IPA (1:1 v/v) with 1% NH4Ac (1M) and HCOOH (0.1%) added. The gradient was programmed as follows: 0 to 2 min 35-80% B, 2 to 7 min 80-100 % B, 7 to 14 min 100% B. The column was equilibrated with a 7min period of 35 % B prior to the next run. The mass spectrometer was a Sciex 5500 QTRAP (Sciex) set in scheduled MRM mode. All lipids were identified for their fatty acid composition by MS/MS to confirm their exact identification, there was also a linear relationship between the increasing number of carbons in the lipid chain and its corresponding retention time. Due to the isobaric nature of sugars we were unable to differentiate Glc and Glc head groups. All data were integrated using the quantitation tool in MultiQuant (3.0.3), all peaks were manually checked. Any analytes which were over the concentration of the standard curve were diluted (1:25) with the same extraction solvent minus the internal standards. The quantification was performed using class-based internal standards and in the case of those ceramide species without an authentic standard in the standard curve mix, we used the closest related structure. The standard curve mixture contained: Glucosyl (beta) C12 ceramide, Lactosyl (beta)) C12 ceramide, C18 ceramide (D18:1/18:1), C18:1 dihydroceramide (d18:0/18:1(9Z)) and was run at the following levels (all in ppb): 100, 80, 60, 50, 40, 30, 20, 10 for the C12 standards and 10, 8, 6, 5, 4, 3, 2,1 for all C18 standards. |
Ion Mode: | POSITIVE |