gender: female tissue: visceral white adipose tissue (vWAT) cell type: T lymphocytes /CD3+ health status: Obese individual: Individual 5 batch: 2
Treatment protocol
vWAT and sWAT samples (n = 22 in total) were obtained from these 11 patients from Cohort 1 during bariatric surgery. Fresh WAT collected during surgeries was transferred into two 50 mL tubes containing a 2% fetal bovine serum (FBS) in phosphate saline buffer (PBS) solution and placed on separate multiwell plate wells submerged in collagenase and Hank's Balanced Salt Solution (pH=7.1). The tissue was then minced into small pieces followed by several centrifugation and supernatant removal steps as previously published (19,20). CD3+ cells were then labeled with anti-CD3+ MicroBeads and magnetically separated from the unlabeled cells by a magnetic-activated cell sorting (MACS) Column supplied by Miltenyi Biotec (Miltenyi Biotec S.L Madrid). Briefly, the cell suspension was centrifuged at 300 x g for 10 min and the supernatant was completely discarded. Second, the pellet was resuspended in 82 µL of MACS buffer and 2 µL were diluted by adding 18 µl of MACS buffer and reserved at 4º C to evaluate percentage of CD3+ cells. Then, 20 µL of CD3+ MicroBeads were added to 80 µL of cell suspension, mixed and incubated for 15 min in ice. Next, the cells were washed by adding 2 mL of MACS buffer and centrifugated at 300 g for 10 minutes at 4ºC. Then the pellet was resuspended in 500 µL of MACS buffer and maintained in ice. Meanwhile, the separation column was prepared. For this, the LS column was placed in the magnetic field, on the MidiMACS™ Separator and rinsed with 3 mL of MACS buffer. Once the column reservoir was empty, the cell suspension was added into the column. When all the suspension passed, the column was washed by adding 3 mL of MACS buffer, twice. After that, the column was removed from the magnetic field, and placed onto a new 15 mL conical tube (collection tube). Then, 5 mL of MACS buffer was added and the fraction with the magnetically labeled cells was immediately flushed out applying the plunger supplied with the column. 20 µL of the eluted fraction was reserved in ice. Next, the CD3+ fraction of cells was centrifuged at 300 g for 7 min at 4º C, 4 mL of the supernatant was discarded, and the pellet was resuspended in the remaining volume, which was transferred into 1.5 mL conical tube and centrifuged at 11000g for 5 min, the resulting pellet was used to perform RNA extraction. The reserved aliquots from sWAT and vWAT were labeled to quantify the viability and the number of infiltrated CD3+ cells, respectively. For this purpose, 10 µL of cell sample were mixed with 48 µL of MACS Buffer, 2 µL of 7-AAD and 0.5 µL of CD3+, the prepared mix was incubated in darkness for 10 min and then 10 µL of Counting beads were added. Isolated T-cells were detected by the flow cytometer FACSCanto II by BD Biosciences. The optic consists of an excitation source and a three-laser system: blue (488 nm, air-cooled, 20-mV solid state), red (633 nm, 17-mV HeNe) and violet (405 nm, 30-mV solid state) allowing measurement of 8 parameters (FSC, SSC and 6 fluorescence detectors) with carrousel acquisition option for tubes. For quantification analysis the FACSDiva software were used, and FlowJo Software (Tree Star Inc.) was used for the graphs and the gating strategy. Cells are being separated based on their size and fluorescence. Viability, percent, and concentration (cells/μL) of T-cells (CD3+) in each tissue (vWAT and sWAT) was measured at the flow cytometer.
Growth protocol
The study was approved by the Ethical Committee of the Hospital Germans Trias i Pujol (Badalona, Spain) and follow the guidelines of Helsinki convention. Participants of both Cohorts 1 and 2 gave their written informed consent before clinical data collection.Two different cohorts of patients were enrolled in this study. All patients were evaluated by the same endocrinologist (S.P.) following the institutional protocol for bariatric surgery between October 2015 and September 2021, according to criteria for bariatric surgery formulated in Spanish Position Statement between Obesity, Endocrinology, Diabetes and Surgery Societies. For Cohort 1, 11 patients with obesity (BMI>35 kg/m2) and different metabolic status were enrolled, and vWAT and sWAT from the same patient were collected on occasion of bariatric surgery. For Cohort 2, 27 patients with or without severe obesity (BMI>35 kg/m2 or BMI<27 kg/m2, respectively) were enrolled; for individuals with obesity, they attended to bariatric surgery, while for normal-weight individuals on occasion of consultation/minor surgery. Then, vWAT and sWAT were collected during the surgical procedures. The inclusion criterion for individuals with obesity was exhibiting a BMI higher than 35 kg/m2. Exclusion criteria for all cohorts were having active infectious or inflammatory pathologies other than those related to obesity and treatment with immunosuppressant drugs, since these conditions could alter the results of the study.
Extracted molecule
total RNA
Extraction protocol
Total RNA was then extracted using affinity column-based methodology suitable for small amounts of biological material (NucleoSpin RNA XS; Mecherey-Nagel, Duren, Germany). RNA yield and purity were determined by spectrophotometry and RNA integrities were assessed with the Nano 6000 assay on Agilent's 2100 Bioanalyzer. Samples with RNA integrity number (RIN) values mostly above 6.7 were used as input for subsequent microarray gene expression profiling assays.
Label
biotin
Label protocol
CD3+ cell extracted RNA was reverse transcribed into cDNA and amplified and biotinylated by in vitro transcription (MultiScribe TaqMan Reverse Transcription Reagents; ThermoFisher Scientific, Waltham, MA, USA).
Hybridization protocol
Labeled cDNAs were hybridized onto Clariom D Human Assay Microarrays, which include probe sets enabling transcriptome-wide gene- and exon-level expression profiling (Affymetrix, ThermoFisher Scientific).
Scan protocol
The arrays were scanned using the GeneChip 3000 system (Affymetrix).
Description
VIS.5
Data processing
Transcript Analysis Console (TAC v4.0; Affymetrix, ThermoFisher) software was used for initial hybridization quality assessment and data inspection. All statistical analyses of microarray data were performed using R-based software environment. Quality control was done with the array QualityMetrics package. Background correction, probe set summarization and normalization were performed with the oligo package using the most up to date annotation in Bioconductor. Subsequent differential expression analysis was performed using the Limma package at the gene level taking the average of all probe sets for a particular gene and focusing on known genes (with assigned gene symbols).
