RNA-Sequencing was performed by the UCLA Technology Center for Genomics & Bioinformatics on endothelial cells isolated from mouse placentas. Briefly, 5 placentas/dam were pooled, and endothelial cells were isolated by magnetic separation using the MidiMACS system following the manufacturer’s instructions (Miltenyi #130-095-927, #130-097-418). Total RNA was extracted using a RNeasy Micro kit (Qiagen) following the manufacturer’s instructions. Expression data were analyzed by comparing cells from the following groups of mice: iron adequate (WT) and iron loaded (HKO) pregnant mice injected on E15.5 with solvent or 0.5 µg/g LPS for 6 h (n = 4 dams/group). Data were sequenced on Illumina HiSeq 3000 for a single-read 50 bp run, and depth of coverage was 2 lanes for >30 million reads/sample. Partek flow and Ingenuity Pathway Analysis (IPA) were used for bioinformatics methods and data analysis, respectively. The reads were mapped to the latest UCSC transcript set using STAR-2.6.1d. The Partek E/M was used to quantify reads to an annotation model. After obtaining gene counts, the counts were normalized by TPM. The Principal Component Analysis was applied to the transcript counts. The differential gene expressions were examined using DESeq2 algorithm. Canonical pathway analysis was performed by IPA using significantly differentially expressed genes (genes filtered by P < 0.05).
Midimacs system
Manufactured by Miltenyi Biotec
Sourced in Spain, Germany
The MidiMACS system is a magnetic cell separation instrument designed for the isolation of target cells from complex biological samples. The system utilizes magnetic beads coated with antibodies or other ligands to specifically label the cells of interest, which can then be separated using a magnetic field. The MidiMACS system is capable of handling larger sample volumes compared to other MACS instruments, making it suitable for a wide range of applications in the field of cell and molecular biology.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2 flow cytometer, by BD (5 mentions)
Facsdiva software, by BD (5 mentions)
Ficoll histopaque, by Merck Group (5 mentions)
K3edta containing tubes, by BD (4 mentions)
Trypan blue exclusion, by Merck Group (2 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
Hiseq 3000, by Illumina (1 mentions)
Na ve cd4 t cell isolation kit, by Miltenyi Biotec (1 mentions)
Rpmi 1640 medium, by Merck Group (1 mentions)
Cd138 microbeads, by Miltenyi Biotec (1 mentions)
Ficoll, by Merck Group (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Ficoll histopaque gradient, by Merck Group (1 mentions)
Labeled cd4 l3t4 mouse microbeads, by Miltenyi Biotec (1 mentions)
Gentlemacs dissociator, by Miltenyi Biotec (1 mentions)
5 bromo 2 deoxy uridine brdu cell proliferation assay, by Roche (1 mentions)
Dextran sedimentation, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by PAN Biotech (1 mentions)
Gelatin solution, by Merck Group (1 mentions)
Amphotericin b, by Merck Group (1 mentions)
23 protocols using midimacs system
1
Placental Endothelial Cell Transcriptomics
2
Isolation of Naive CD4+ T Cells
3
Isolation and In Vitro Treatment of Monocytes
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This study was managed according to the Good Clinical Practice Guidelines and in line with the principles delimited in the Helsinki Declaration of the World Medical Association. Blood was obtained from healthy donors in Centro Regional de Transfusion Sanguinea de Sevilla-Huelva y Banco de Tejidos. Firstly, the peripheral blood mononuclear cells (PBMCs) were isolated by centrifugation over a Ficoll-Histopaque (Sigma-Aldrich, Madrid, Spain) gradient from buffy coat. CD14 microbeads and LS columns on a midiMACS system (Miltenyi Biotec, Madrid, Spain) were used in order to isolate monocytes from PBMCs according to the specified protocol of the manufacturer. The purity for CD14 monocyte isolations was routinely >95% by flow cytometry (FACScanto II flow cytometer and FACSDiva software, versión 5.0.1., Becton Dickinson, Erembodegem, Belgium) [30 (link)]. After that, monocytes were cultivated in a supplemented (L-glutamine, 1% penicilin/streptomicin, 10% foetal bovine serum) Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma-Aldrich, St. Louis, MO, USA). Finally, the in vitro treatments were performed over purified monocytes in a density of 5 × 105 in a 12-well plate. Monocytes were previously stimulated with or without 0.1 µL/mL of LPS, and were exposed to WGPH45A for 24 h at 50 and 100 µg/mL.
4
Isolation of Multiple Myeloma Cells
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This study was approved by the University of South Florida Institutional Review Board protocol. Written informed consent for the use of BM aspirates was obtained from all patients. BM samples were collected from patients with MM. BM mononuclear cells were isolated using Ficoll-Paque gradient centrifugation and incubated with CD138-MicroBeads followed by magnetic separation of positive cells using MidiMACS system according to the manufacturer protocol (Miltenyi Biotec Inc, San Diego, CA). Cells were cultured in -minimal essential medium supplemented with 10% fetal bovine serum and 1% antibiotic/antimycotic. Bone marrow stroma (BMS) was generated from BM mononuclear cells as described earlier [13 (link)].
5
Isolation and Stimulation of Human Monocytes
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Peripheral blood mononuclear cells (PBMCs) were obtained from buffy coats donated by Centro Regional de Transfusiones Sanguíneas y Banco de Tejidos de la provincia de Sevilla y Huelva. PBMCs were isolated by centrifugation on a gradient with Ficoll (Sigma, Madrid, Spain) [31 (link)] and monocytes were separated from PBMCs by positive selection using CD14 microbeads and LS columns on a midiMACS system (Miltenyi Biotec, Madrid, Spain) according to the manufacturer’s instructions. After isolation, monocytes were suspended in RPMI 1640 medium supplemented with L-glutamine, penicillin, streptomycin, and 1% heat-inactivated fetal bovine serum. For treatments, 5 × 105 monocytes/well were seeded in 24-well plates in presence or absence of LPS (100 ng/mL) (Sigma, Madrid, Spain) and treated with CPH at 50 and 100 μg/mL for 24 h.
6
Isolation of Monocytes from Peripheral Blood
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The same six volunteers who took part as donors of postprandial TRLs participated as donors of monocytes. After an overnight fasting period of 12 h, peripheral blood samples were drawn from a large antecubital vein and collected into K 3 EDTA-containing tubes (BD). Peripheral blood mononuclear cells (PBMCs) were isolated by centrifugation over a Ficoll-Histopaque (Sigma) gradient. Monocytes were isolated from PBMCs using CD14 microbeads and LS columns on a midiMACS system (Miltenyi Biotec). Monocytes were tested for purity by CD14 fluorescein isothiocyanate (FITC) labelling and fluorescence-activated cell sorter (FACS) analysis. The detailed method can be found in the Supporting Information.
7
Profiling Plasma Cell Dyscrasia
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PCs were purified from BM samples using CD138 immunomagnetic microbeads (MidiMACS system, Miltenyi Biotec, Auburn, CA) and the purity of the positively selected PCs was >90% in all cases. Gene expression profiles (GEP) were investigated in a panel of 268 patients included in two different datasets and representative of all the major forms of PC dyscrasia: a proprietary dataset at NCBI Gene Expression Omnibus repository (accession #GSE66293) previously profiled by us (4 normal controls and 129 MM patients)13 (link),14 (link); and a publicly available data set including five normal controls, 20 MGUS, 33 SMM, and 41 MM patients (accession #GSE47552)15 (link). The cohort consists of newly-diagnosed patients. The proprietary 129 MM tumors (accession #GSE66293) employed for the study were representative of the major molecular characteristics of the disease and stratified according the TC classification16 (link),17 (link). Deletions of 17p13, 13q14 and gain of 1q were also evaluated by FISH18 (link). Procedures followed the rules indicated in the declaration of Helsinki. The internal Institutional Board approved the use of human material.
8
Isolation and Culture of Human Monocytes
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The “Centro Regional de Transfusiones Sanguíneas y Banco de Tejidos de la province de Sevilla y Huelva” provided samples from which peripheral blood mononuclear cells (PBMC) were obtained from buffy coats after centrifugation using a Ficoll-Histopaque gradient (Sigma). According to the manufacturer’s instructions, with a midiMACS system (Miltenyi Biotec, Madrid, Spain), monocyte cells were isolated from PBMC using CD14 microbeads and LS columns. By flow cytometry (FACScanto II flow cytometer and FACSDiva software, BD), the purity for CD14 monocyte isolations was quantified routinely at >90%. After isolation, monocyte cells became resuspended in RPMI 1640 medium, in which 10% heat-inactivated fetal bovine serum, penicillin, streptomycin, and L-glutamine were added [34 (link)].
9
Isolation of Primary Human Myeloma Cells
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Primary human MM cells were recovered from fresh BM aspirates of MM patients. The hPBMCs were isolated by Ficoll density gradient centrifugation. CD138 positive cells were selected by magnetic bead sorting using the MidiMACS system (Miltenyi Biotec Inc., Bergisch Gladbach, Germany) according to the manufacturer’s instructions.
10
T Cell Proliferation Assay for Pmp18.1
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We used the DC-naïve T cell proliferation assay to evaluate the ability of Pmp18.1 to elicit helper CD4+ T cell proliferation. Splenocytes obtained from spleens of naïve mice using the gentleMACS Dissociator (Miltenyi Biotech, Auburn, CA) were resuspended in PBS containing BSA and EDTA. Purification of CD4+ T cells was achieved by positive selection using the MidiMACS system and labeled CD4 (L3T4) mouse microbeads (Miltenyi Biotech, Auburn, CA). Purity of the CD4+ T cells was analyzed by flow cytometry and shown to be at least >95%. Purified naïve CD4+ T cells (2 × 105 cells/well) were cultured in 96-well plates (Corning Glass Work, Acton, MA) with BMDCs (2 × 105 cells/well) and either Pmp18.1 (10 μg/ml) or Pmp18.1 plus VCG (100 μg) in 200 μl of C-RPMI medium supplemented with GM-CSF at 37°C in 5% CO2. Control cultures containing DCs and naïve T cells without antigen served as internal control. After 7 days of culture, T cell proliferation was assessed essentially as described previously (Eko et al., 2011a (link)) using the 5-Bromo-2′-deoxy-uridine (BrdU) cell proliferation assay (Roche Molecular Biochemicals, Indianapolis, IN). Absorbance values were read at 450 nm and the ratio between stimulated and non-stimulated cells (stimulation index, SI) was then calculated.
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