At the end of the co-culture, total cells were resuspended at 107 cells per 100 µl in cold PBS (4°C) supplemented with 0.1% Bovine Serum Albumin (BSA, Sigma–Aldrich) and 2 mM EDTA (VWR, Fontenay-sous-Bois, France). Phyco-Erythrin (PE)-coupled Pro5 MHC class I Pentamer expressing M1m (Pentamer M1m, Proimmune, Oxford, UK) was added in medium (100 ng for 107 cells) for 30 min. Cells were then washed and incubated with anti-PE microbeads (Miltenyi Biotec) for 15 min and finally washed again and resuspended in 500 µl of cold PBS supplemented with 0.1% BSA and 2 mM EDTA. During the different steps of purification, cells were always stored on ice and all reagents were kept at 4°C. Magnetic sorting was realized following manufacturer's protocol. Cells were amplified on AAPCs (5 × 105 T cells on 105 irradiated AAPCs), as described above for 14 days, and then used in both phenotypic and functional studies at D35.
Ethylene diamine tetraacetic acid (edta)
Manufactured by Miltenyi Biotec
Sourced in Germany
EDTA is a versatile laboratory reagent used in various applications. It functions as a chelating agent, capable of binding to metal ions and disrupting their chemical interactions. This property makes EDTA useful in applications that require the removal or sequestration of certain metal ions from solutions.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Merck Group (5 mentions)
Bovine serum albumin (bsa), by Miltenyi Biotec (4 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (3 mentions)
Cell strainer, by Miltenyi Biotec (2 mentions)
Dpbs 1x, by Miltenyi Biotec (2 mentions)
Dpbs 1x, by Merck Group (2 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (2 mentions)
Dpbs 1x, by Thermo Fisher Scientific (2 mentions)
Facscalibur, by BD (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Ethylene diamine tetraacetic acid (edta), by Avantor (1 mentions)
Anti pe microbeads, by Miltenyi Biotec (1 mentions)
Nylon strainer, by BD (1 mentions)
Interleukin 2 (il 2), by Merck Group (1 mentions)
Collagenase d, by Worthington (1 mentions)
Human serum, by Merck Group (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
Gentlemacs, by Miltenyi Biotec (1 mentions)
7 aad staining solution, by Miltenyi Biotec (1 mentions)
Mhcii apc, by Miltenyi Biotec (1 mentions)
12 protocols using ethylene diamine tetraacetic acid (edta)
1
Antigen-Specific T Cell Enrichment
2
Flow Cytometry Analysis of HUVEC Cells
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HUVEC cells grown in 6-well plates and treated with DMSO, PTC, CRT0066101 (5 µM) or Kb-NB-142-70 (5 µM) as indicated. Cells were washed with PBS and detached by PBS supplemented with EDTA (Sigma–Aldrich) for 3 min. The cells were resuspended in FACS buffer (PBS, 5% FCS, 0.05% EDTA) that contained the fluorescently labelled primary-antibodies (Miltenyi Biotec). Staining was performed on ice for 1 h. Flow cytometry was done using an LSRII special order FACS System (BD Bioscience) or an Attune NxT analytical flow cytometer (Thermo Scientific). Data was evaluated and plotted with Flowjo (Agilent).
3
Isolation of Human and Murine Lamina Propria Mononuclear Cells
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Human LPMC [lamina propria mononuclear cells] were isolated as previously described.58 (link) Murine LPMC were isolated as in Burrello et al.23 (link) Briefly, the dissected human intestinal mucosa was freed of mucus and epithelial cells in sequential steps with DTT [0.1 mmol/L] and EDTA [1 mmol/L] [both from Sigma] and then digested with collagenase D [400 U/ml] [Worthington Biochemical Corporation, Lakewood, NJ] for 5 h at 37°. LPMCs were then separated with a Percoll gradient and cultured in complete RPMI 1640 medium containing 5% human serum [Sigma] and 100 UI/ml IL-2 [Proleukin].
For murine LPMC isolation, colonic lamina propria mononuclear cells were isolated via incubation with 5 mM EDTA at 37°C for 30 min, followed by mechanical disruption with GentleMACS [Miltenyi Biotec]. After filtration with 100-µm and 70-µm nylon strainers [BD], the LPMC were counted and stained for immunophenotyping.
For murine LPMC isolation, colonic lamina propria mononuclear cells were isolated via incubation with 5 mM EDTA at 37°C for 30 min, followed by mechanical disruption with GentleMACS [Miltenyi Biotec]. After filtration with 100-µm and 70-µm nylon strainers [BD], the LPMC were counted and stained for immunophenotyping.
4
Isolation and Characterization of Colonic Immune Cells
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The intestines of 16-week-old Winnie and WT mice were removed and tissue digested to obtain a single-cell preparation. Colons were cut into small segments (1 cm long), washed with DPBS 1X (Gibco, Waltham, MA, USA) + 2.5 mM EDTA (Ambion, Thermo Fisher Scientific, Waltham, MA, USA) to remove epithelial cells, and digested with collagenase type IV and DNase I (Sigma Aldrich, St. Louis, MO, USA) using the GentleMacs suggested protocol for 30 minutes at 37°C. Resulting single-cell suspensions from colonic lamina propria (LP) were pelleted by centrifugation, washed with DPBS 1X + 0.5 mM EDTA, and passed through 100-μm and 30-μm cell strainers (Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were then washed with DPBS 1X + 0.5% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO, USA) and labeled with CD45.2-FITC and MHC II-APC (Miltenyi Biotec, Bergisch Gladbach, Germany), according to the manufacturer’s instructions. Finally, cell suspensions were labeled with 7-AAD Staining Solution (Miltenyi Biotec, Bergisch Gladbach, Germany) to distinguish between viable and dead cells, also according to the manufacturer’s instructions. Flow cytometer data analysis was performed using NAVIOS software (Beckman Coulter, Brea, CA, USA), with at least 3 experiments performed.
5
Monocyte Isolation from Tissues
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Cell sorting of monocytes bulks were preceded by a positive selection of CD11b+ cells from SILP, spleen, or BM cells resuspended in PBS with 2% FCS (Lonza) and 100 mM EDTA, CD11b MicroBeads, human and mouse (Miltenyi biotec), and Fc receptor-blocking antibodies for 20 min in the dark at 4 °C. Cell separation was performed on the auto-MACS-system according to the manufacturer's recommendations (Miltenyi Biotec). The positive fraction was incubated with Fc receptor-blocking antibodies and a surface staining antibody mix (see Flow cytometry). Single cells and bulks were sorted on a BD FACSAria™ III (see Supplementary Fig. 1 for gating strategy).
6
Bone Marrow Cell Surface Marker Analysis
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Bone marrow cells were analyzed for the surface expression of PE CXCR4 (L276F12; Biolegend), PE β1 integrin (HM B1-1; BD Pharmingen), PE α4 integrin (9C10; BD Pharmingen), PE-α6 integrin (GoH3; BD Pharmingen), and PE-CD44 (IM7; BD Pharmingen) on the LSK population. In addition, we also assessed Rac-1 (Cytoskeleton) surface expression. Isolated bone marrow cells were treated with Fc block prior to staining with the LSK markers. All samples were labeled in MACs buffer (phosphate-buffered saline [PBS], 0.5% bovine serum albumin [BSA], 2 mM EDTA, pH 7.2; Miltenyi Biotec) for 30 min on ice. Samples were washed three times with MACs buffer after staining and analyzed using the LSR Fortessa (BD Bioscience). Histograms were created using FlowJo software.
7
hiPSCs-MSCs Phenotypic Characterization
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The hiPSCs-MSCs at passage 3 were harvested. One million cells were suspended in 100 µl buffer that consisted of 0.5% bovine serum albumin (BSA; Sigma-Aldrich; Merck Millipore) and 2 mM EDTA (Sunshine Biotechnology Co., Ltd., Nanjing, China). Subsequently, 10 µl 1:10 diluted fluorescein isothiocyanate (FITC)-coupled antibodies recognizing CD11b (130-098-778), CD105 (130-098-778), CD90 (130-097-930), CD45 (130-098-043) and CD34 (130-098-142) (MACS; Miltenyi Biotec, Bergisch Gladbach, Germany) were added. In addition, 1:10 diluted mouse IgG1 (130-104-562) and mouse IgG2a (no. 130-098-877) antibodies (MACS; Miltenyi Biotec) were used as isotype controls. Incubation for 10 min incubation in the dark at 4°C was performed. The cells were then washed with buffer containing phosphate-buffered saline, pH 7.2, 0.5% BSA, and 2 mM EDTA by diluting MACS BSA Stock Solution (130-091-376) 1:20 with autoMACS Rinsing Solution (130-091-222) (MACS; Miltenyi Biotec). Then, the cells were centrifuged at 300 × g for 10 min at 4°C and resuspended in 500 µl of the aforementioned buffer for analysis by flow cytometry (BD FACSCalibur, BD Biosciences, Franklin Lakes, NJ, USA). The data was analyzed using Flowjo 7.6 sofrware (BD Biosciences).
8
Isolation of T Cell Subsets
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Following respirometry, cell suspensions were centrifuged and PBMC were resuspended in cell separation buffer (PBS, 0.5% bovine serum albumin, 2 mM EDTA; Miltenyi Biotec, Bergisch-Gladbach, Germany) for the separation of T cell subsets. Appropriate volumes of antibodies (CD4-APC [10 µl/107 cells], CD8-FITC [10 µl/107 cells], CD45RA-PE [5 µl/107 cells], CD3-PE-Vio770 [5 µl/107 cells], all purchased from Miltenyi Biotec) were added and cells were stained according to the manufacturer’s protocol. Naïve T helper cells (CD3+CD4+CD45RA+), memory T helper cells (CD3+CD4+CD45RA−), naïve cytotoxic T cells (CD3+CD8+CD45RA+), and memory cytotoxic T cells (CD3+CD8+CD45RA−) were then isolated by fluorescent-activated cell sorting on a BD FACSAria III cell sorter (BD Biosciences, Heidelberg, Germany). Propidium iodide staining was used to distinguish dead from living cells.
9
Purification and Culture of Mouse Retinal Ganglion Cells
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Mouse RGCs were purified using a magnetic-bead separation method that has been previously described.18 (link) Briefly, P0 to P5 mouse retinas from CD1 mice were dissected in Mg2+/Ca2+-free Hanks' balanced salt solution (HBSS). Retinas were then digested for 5 minutes at 37°C in HBSS containing 20 U/mL papain and 0.005% DNase I (Worthington Biochemicals, Lakewood, NJ, USA). Digestion was neutralized with ovomucoid and 0.005% DNase I (Worthington Biochemicals) and then the retina was triturated with a pipette. Dissociated cells were treated with rabbit anti-mouse Thy1.2 antibody conjugated to micrometal beads (130-049-101; Miltenyi Biotech, Auburn, CA, USA) for 15 minutes at room temperature in elution buffer (phosphate-buffered saline with 0.5% bovine serum albumin and 2 mM EDTA; Miltenyi Biotech). RGCs were then purified from the cell suspensions using a metal column in the presence and absence of a magnetic field. Purified RGCs were quantified and then diluted to 0.5 × 106 RGCs/mL in medium (see above). Cells (1 mL) were then plated onto an electrotaxis chamber (see below) and placed in incubator at 37°C overnight for 12 hours.
10
Genotyping of Healthy Volunteers
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Twenty-three healthy volunteers were recruited at the University of Manchester as part of the National Repository Healthy Volunteers (NRHV) study. All the samples were collected with ethical committee approval (MREC 99/8/84) and all individuals provided informed consent. The median age of the subjects was 50.5 years (range=26–82 years) with eight males and 15 females. A total of 20 ml peripheral blood was collected in Vacutainer plus tubes containing EDTA (Becton Dickinson). PBMCs were extracted within 2 h of collection from 20 ml of whole blood using Ficoll Plus density gradient centrifugation (GE Healthcare). Cells were washed twice with MACS running buffer (phosphate-buffered saline (PBS), bovine serum albumin, EDTA and sodium azide; Miltenyi) and total PBMCs counted using the CASY cell counter (Roche). To allow collection of all the samples, PBMC samples were immediately cryopreserved in 1 ml recovery cell-freezing medium (Gibco) per 5 × 106 cells at a cooling rate of −1 °C per minute. Samples were genotyped using the Illumina HumanCoreExome v1.0 array, in accordance with the manufacturer’s instructions. Genotype clustering and calling was performed using the GenomeStudio Data Analysis software platform using the Illumina HumanCoreExome-24 v1.0 Manifest File.
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