YFP+ Treg cells were isolated by sorting lymph node samples pre-enriched for CD25+ cells using a mouse CD25 Microbead kit (Miltenyi Biotec) on a FACSAria (BD Biosciences). CD4+CD25- Tconv cells were isolated from the CD25- fraction obtained from the same samples by negative selection using FITC-conjugated antibodies and anti-FITC Microbeads (Miltenyi Biotec) as described above (T cell isolation). Tregs were co-cultured in known ratios with 1x105 Tconv cells per well in 96-well round-bottom Nunclon plates (Nunc) and stimulated with 2x104 anti-CD3/anti-CD28-coated Dyna beads (Dynal). After 96 hours’ incubation at 37°C in an atmosphere of 5% CO2, proliferation was measured by [3H]-thymidine incorporation. [3H]-thymidine was added at 0.5μl per well and plates incubated for 6 hours before cells were harvested to UniFilter plates (Perkin Elmer) using a Tomtec 96 Harvester. Collection plates were air-dried overnight and 30μl MicroScint™-20 (Perkin Elmer) added to each well. A TopcountTM scintillation counter (Perkin Elmer) was then used to read the relative incorporation of [3H]-thymidine in each well.
Nunclon plate
Manufactured by Thermo Fisher Scientific
Sourced in Denmark, United States
Nunclon plates are a range of cell culture plates designed for adherent cell growth. The plates feature a surface treatment that promotes cell attachment and proliferation. They are available in various formats and well counts to accommodate diverse experimental needs.
Automatically generated - may contain errors
Lab products found in correlation
Glutamax, by Thermo Fisher Scientific (4 mentions)
Neurobasal medium, by Thermo Fisher Scientific (3 mentions)
Microscint 20, by PerkinElmer (2 mentions)
Mouse cd25 microbead kit, by Miltenyi Biotec (2 mentions)
Anti cd3 anti cd28 coated dyna beads, by Thermo Fisher Scientific (2 mentions)
Unifilter plates, by PerkinElmer (2 mentions)
Facsaria, by BD (2 mentions)
Anti fitc microbeads, by Miltenyi Biotec (2 mentions)
Polyornithine laminin, by Thermo Fisher Scientific (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Topcount scintillation counter, by PerkinElmer (1 mentions)
Laminin, by Thermo Fisher Scientific (1 mentions)
Horse serum, by Vector Laboratories (1 mentions)
Nerve growth factor (ngf), by Merck Group (1 mentions)
Poly d lysine pdl, by Merck Group (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Spectramax m5e multi mode microplate reader, by Molecular Devices (1 mentions)
Cpg odn 2006, by Miltenyi Biotec (1 mentions)
Ionomyocin, by Merck Group (1 mentions)
13 protocols using nunclon plate
1
Treg Suppression Assay: Quantifying Regulatory T Cell Function
2
Isolation and Stimulation of hPBMCs
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For hPBMC isolation, human blood was collected from healthy volunteers according to the local guidelines and regulations, as approved by the College Ethics Review Board of the University of Aberdeen (CERB/2012/11/676 and CERB/2016/8/1300). The isolation of hPBMCs was essentially performed as previously described (Endres et al., 1988 (link)). Samples of 5*105 freshly isolated hPBMCs were placed into round-bottom 96-well Nunclon plates (Nunc, Roskilde, Denmark) and stimulated with heat-killed 2*105 yeast cells for 24 h at 37 °C and 5% (v/v) CO2. For the preparation of glucan-phosphate-treated human PBMCs, cells were incubated with 10.0 µg of glucan-phosphate (kind gift from Prof. David L Williams) for 2 h at 37 °C and 5% (v/v) CO2 before challenge with fungal cells. Additionally, for understanding the temperature-induced cell wall changes in wild type strains, the hPBMCs were similarly challenged with thimerosal killed fungal cells exposed to different temperatures, as described in the material and methods section. The supernatants were collected after 24 h of challenge and stored at −20 °C till cytokine analysis was performed. Cytokine measurements were carried out by ELISA using commercially available kits from R&D systems (Abingdon, UK).
3
Isolation and Culture of Murine Spinal Motor Neurons
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Murine embryonic spinal MNs were isolated and cultured as previously described (Wiese et al., 2010 (link)). Briefly, after dissection of the ventrolateral part of E12.5 embryos, spinal cord tissues were incubated for 15 min in 0.05% trypsin in HBSS. Cells were triturated and incubated in Neurobasal medium (Invitrogen), supplemented with 1× Glutamax (Invitrogen) on Nunclon plates (Nunc) precoated with antibodies against the p75 NGF receptor (MLR2; kind gift of Robert Rush, Flinders University, Adelaide, Australia) for 45 min. Plates were washed with Neurobasal medium, and the remaining MNs were recovered from the plate with depolarization solution (0.8% NaCl, 35 mM KCl and 2 mM CaCl2) and collected in full medium (2% horse serum and 1× B27 in Neurobasal medium with 1× Glutamax). After counting, the cell number was adjusted to 1,000 in 100 µl, and 1,000 cells were plated per well on four-well dishes (Greiner; Cellstar) precoated with poly-ornithine/laminin (Invitrogen). Cells were cultured in the presence of brain-derived neurotrophic factor. Axon length was quantified after 7 d in vitro.
4
Isolation and Culture of Murine Embryonic Spinal Motoneurons
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Murine embryonic spinal motoneurons were isolated and cultured as described56 (link). Briefly, after dissection of the ventrolateral part of E12.5 embryos, spinal cord tissues were incubated for 15 min in 0.05% trypsin in Hank’s balanced salt solution. Cells were triturated and incubated in Neurobasal medium (Invitrogen), supplemented with 1× Glutamax (Invitrogen) on Nunclon plates (Nunc) pre-coated with antibodies against the p75 NGF receptor (MLR2, kind gift of Robert Rush, Flinders University, Adelaide, Australia) for 45 min. Plates were washed with Neurobasal medium, and the remaining motoneurons were recovered from the plate with depolarization solution (0.8% NaCl, 35 mM KCl and 2 mM CaCl2) and collected in full medium (2% horse serum, 1× B27 in Neurobasal medium with 1× Glutamax). After counting, cell number was adjusted to 1000 in 100 µl, and 1000 cells were plated on four-well dishes (Greiner, Cellstar) pre-coated with poly-ornithine/laminin (Invitrogen). Cells were cultured in the presence of the neurotrophic factor BDNF. For survival assays, cells were counted 4 h after plating to find the total number of plated cells. Cells were counted again after 5 and after 7 days in vitro (DIV).
For lentiviral transduction, motoneurons were incubated with viral particles for 10 min at RT directly before plating.
For lentiviral transduction, motoneurons were incubated with viral particles for 10 min at RT directly before plating.
5
Suppressive Capacity of Regulatory T Cells
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YFP+ Treg cells were isolated by sorting lymph node samples pre-enriched for CD25+ cells using a mouse CD25 MicroBead Kit (Miltenyi Biotec) on a FACSAria (BD Biosciences). CD4+CD25− Tconv cells were isolated from the CD25− fraction obtained from the same samples by negative selection using FITC-conjugated Abs and anti-FITC Microbeads (Miltenyi Biotec) as described above (T cell isolation ). Treg cells were cocultured in known ratios with 1 × 105 Tconv cells per well in 96-well round-bottom Nunclon plates (Nunc) and stimulated with 2 × 104 anti-CD3/anti-CD28–coated Dyna Beads (Dynal). After 96-h incubation at 37°C in an atmosphere of 5% CO2, proliferation was measured by [3H]thymidine incorporation. [3H]thymidine was added at 0.5 μl per well, and plates were incubated for 6 h before cells were harvested to UNIFILTER Plates (PerkinElmer) using a Tomtec 96 Harvester. Collection plates were air dried overnight and 30 μl MicroScint-20 (PerkinElmer) added to each well. A TopCount Scintillation Counter (PerkinElmer) was then used to read the relative incorporation of [3H]thymidine in each well.
6
Murine Embryonic Spinal Motor Neuron Culture
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Primary MN culture Murine embryonic spinal MNs were isolated and cultured as described (L€ uningschro ¨r et al., 2017) . Briefly, after dissection of the ventrolateral part of E12.5 embryos, spinal cord tissues were incubated for 15 minutes in 0.1% trypsin in Hank's balanced salt solution. Cells were triturated and incubated in Neurobasal medium (Invitrogen, CA, USA), supplemented with 1 3 Glutamax (Invitrogen, CA, USA) on Nunclon plates (Nunc) pre-coated with antibodies against the p75 NGF receptor (MLR2, kind gift of Robert Rush, Flinders University, Adelaide, Australia) for 45 minutes. Plates were washed three times with Neurobasal medium, and the remaining MN were recovered from the plate with depolarization solution (0.8% NaCl, 35 mM KCl and 2 mM CaCl 2 ) and collected in MN medium (2% horse serum, 1x B27 in Neurobasal medium with 1x Glutamax).
7
Culturing Isolated Dorsal Root Ganglia
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DRGs were isolated using previously described protocols (Vogelaar et al. 2009 (link)). Briefly, the spinal column of adult mice was opened via laminectomy and DRGs were pulled out of the intervertebral foramina. After removing nerve stumps and connective tissue, two DRG explants per well were plated in 4-well-Nunclon plates (Thermo scientific) coated with poly-D-lysine (PDL; 0.5 mg/ml; Sigma) and laminin (1 µg/ml; Life Technologies). Growth medium consisted of neurobasal medium containing 2% horse serum (Vector Laboratories), 1% Glutamax, 2% B-27 and 1% penicillin/streptomycin (all Life Technologies), supplemented with 10 ng/ml nerve growth factor (NGF, Sigma). Explants were cultivated for 4–6 days in an incubator at 37 °C with 5% CO2.
8
Fluorescent Oligonucleotide Binding Assay
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hex4_A5U oligonucleotides were labeled with either 5′ 6-carboxyfluorescein (hex4_A5U-5F) or 3′ carboxytetramethylrhodamine (hex4_A5U-3T) or both fluorophores (hex4_A5U-5F3T). Reactions were set up in triplicate in 96-well Nunclon plates (Thermo Fisher Scientific, Waltham, MA) containing 200 nM of either hex4_A5U-5F3T or a mix of 100 nM hex4_A5U-5F + 100 nM hex4_A5U-3T annealed in 10 mM TBA (pH 7.5) + 100 mM KCl or 100 mM LiCl. Protein was added at 0, 200 nM, 500 nM, or 1000 nM concentrations and incubated for 1 h at 4 °C before data collection. Data were collected on a Spectramax M5e Multi-Mode Microplate Reader (Molecular Devices, Sunnyvale, CA, USA) and processing was performed as previously described [33 (link)]. Labeling and data collection for A5UR20 oligonucleotides were done as described for hex4_A5U.
9
Induction of IL-10 Expression in B Cells
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PBMCs were isolated as described earlier. CD4 + T cells and CD19 + B cells were subsequently isolated by magnetic cell sorting, based on CD4 microbead positive selection and subsequent CD19-negative selection (MACS, Miltenyi Biotech); purity was consistently >98%. Cells were cultured in 10% FBS, RPMI-1640 (Sigma) with 2 mM l -glutamine (Sigma), 0.1 g/L sodium bicarbonate (Sigma), supplemented with 100 U/mL penicillin and 0.1 mg/mL Streptomycin (PAA), and 10 mM Hepes (PAA), in round-bottom 96-well Nunclon plates (Thermo Scientific) at 0.3 × 106 cells/well. To induce IL-10 expression, the TLR-9 ligand CpG ODN 2006 (Miltenyi Biotech) was used, alongside IL-2 (250 mcg/ml, Aldesleukin, Peprotech). B cells were cultured for up to 40 h. This time point was chosen as most appropriate for the detection of IL-10, based on kinetics of expression. Cells were re-stimulated with PMA (1 mg/mL) and Ionomyocin (1 mg/mL, Sigma) in the presence of Brefeldin A, and GolgiStop (both 1 mg/ml) for three hours at a 1:1000 concentration, and then stained for intracellular cytokine production.
10
Expansion of Subcutaneous ASCs
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Subcutaneous ASCs, passage 0 (p0), were purchased from Obatala Sciences (New Orleans, LA, USA). The cells were seeded at 400 cells/cm2 in 150 cm2 Nunclon plates (ThermoScientific, Pittsburgh, PA, USA) and expanded with growth medium consisting of Dulbecco’s modified Eagle medium Nutrient Mixture F-12 (Gibco, Gaithersburg, MD, USA), supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA) and 1% antibiotic/antimycotic (anti/anti, Gibco) as previously described [35 (link)].
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