Polyclonal T cell lines were generated by footpad priming mice with emulsions of MOG35-55 or NFM15-35 (1 mg/mL) in CFA containing a final concentration of 0.5 mg/mL heat-killed Mycobacterium tuberculosis (H37 RA Mtb) in Incomplete Freund's Adjuvant (BD). Draining lymph nodes were harvested 12-14 days after priming. Single cell suspensions were generated by passing cells through a 100μm cell strainer and directly assessed for antigen specific proliferation by uptake of [3H] tritiated thymidine (VWR International, Inc). 6×105 primed lymph node cells or naïve 2D2 splenocytes were cultured in a 96-well flat bottom plate with indicated peptides and concentrations. After 48 hours, 3H-thymidine (0.4μCi/well) was added to the culture media for 24 hours before the cells were harvested onto a filtermats (PerkinElmer) with the FilterMate 196 harvester (Packard). 3H-thymidine uptake was analyzed with the 1450 Microbeta TriLux microplate liquid scintillation counter (PerkinElmer). Cell culture media contained RPMI 1640 (CellGro) supplemented with 10% heat-inactivated FBS (Gibco-Life Technologies), 4mM L-glutamine (CellGro), 0.01M HEPES (CellGro), 100μg/mL gentamicin (CellGro), 20μM 2-ME (Sigma-Aldrich). Stimulation index was calculated as a ratio of the counts per minute between peptide stimulated versus unstimulated cells.
Filter mats
Manufactured by PerkinElmer
Sourced in United States
Filter mats are a type of laboratory equipment used for filtration processes. They are made of materials designed to efficiently separate solid particles from liquids or gases. The primary function of filter mats is to provide a reliable and effective means of filtration in various scientific and industrial applications.
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Lab products found in correlation
3h thymidine, by PerkinElmer (4 mentions)
Heat inactivated fbs, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Corning (1 mentions)
Rpmi 1640, by Corning (1 mentions)
Hepes, by Corning (1 mentions)
Filtermate 196 harvester, by Hewlett-Packard (1 mentions)
Gentamicin, by Corning (1 mentions)
Concanavalin a (cona), by Merck Group (1 mentions)
Anti cd3 clone 145 2c11, by BD (1 mentions)
Anti cd28 clone 37, by BD (1 mentions)
3h thymidine, by GE Healthcare (1 mentions)
Interleukin 2 (il 2), by R&D Systems (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Wallac 1450 microbeta trilux liquid scintillation counter, by PerkinElmer (1 mentions)
Aim 5 medium, by Thermo Fisher Scientific (1 mentions)
Mach 3 cell harvester, by PerkinElmer (1 mentions)
Meltilex, by PerkinElmer (1 mentions)
Cd8 rosettesep, by STEMCELL (1 mentions)
6 protocols using filter mats
1
Antigen-Specific T Cell Proliferation Assay
2
Proliferation Assay for Splenocyte Activation
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Proliferation assays were performed as previously described
[15 (link)]. In brief, splenocytes were isolated from C57BL/6 adult mice or fetal sheep and seeded in 96-well, flat-bottom microtiter plates (Nunc, Thermo Fisher Scientific, Australia). Assays were performed in triplicate at a concentration of 2.5 × 105 cells per well in complete RPMI medium alone or in the presence of 5 μl/ml concanavalin A (ConA; Sigma-Aldrich), or into wells precoated with 10 μg/ml anti-CD3 (clone 145-2C11) and 10 μg/ml anti-CD28 (clone 37.51) antibodies (both from BD Biosciences) to a final volume of 200 μl per well. For wells that required addition of hAECs, 50 μl of hAECs that had been exposed to surfactant or PBS, at hAEC-to-splenocyte ratios ranging from 1:5 to 1:40, were added to each well before the addition of splenocytes. Cells were incubated at 37°C for 48 hours and then 1 μCi/well [3H]-thymidine (Perkin Elmer, Waltham, MA, USA) was added for an additional 18 hours of culture. Cells were harvested onto filter mats (Perkin Elmer), and incorporated radioactive nucleic acids were counted by using a Top Count Harvester (Packard Biosciences, Meriden, CT, USA).
[15 (link)]. In brief, splenocytes were isolated from C57BL/6 adult mice or fetal sheep and seeded in 96-well, flat-bottom microtiter plates (Nunc, Thermo Fisher Scientific, Australia). Assays were performed in triplicate at a concentration of 2.5 × 105 cells per well in complete RPMI medium alone or in the presence of 5 μl/ml concanavalin A (ConA; Sigma-Aldrich), or into wells precoated with 10 μg/ml anti-CD3 (clone 145-2C11) and 10 μg/ml anti-CD28 (clone 37.51) antibodies (both from BD Biosciences) to a final volume of 200 μl per well. For wells that required addition of hAECs, 50 μl of hAECs that had been exposed to surfactant or PBS, at hAEC-to-splenocyte ratios ranging from 1:5 to 1:40, were added to each well before the addition of splenocytes. Cells were incubated at 37°C for 48 hours and then 1 μCi/well [3H]-thymidine (Perkin Elmer, Waltham, MA, USA) was added for an additional 18 hours of culture. Cells were harvested onto filter mats (Perkin Elmer), and incorporated radioactive nucleic acids were counted by using a Top Count Harvester (Packard Biosciences, Meriden, CT, USA).
3
PBMC Proliferation Assay with Irradiated Tumor Cells
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Peripheral blood mononuclear cells (PBMCs) were freshly isolated from healthy volunteers who had given informed consent after full ethical approval (Cardiff University Biobank; www.cardiff.ac.uk/biobank ). PBMCs were isolated from the buffy coat following density gradient centrifugation on Histopaque. Isolated PBMCs were resuspended and washed 3 times in RPMI medium containing 10% (v/v) FBS to remove any platelets or contaminating histopaque.
As part of the assay, to prevent the OMLP-PC cell lines from proliferating, they were irradiated with 20 Gy (iOMLP-PCL). 1 × 105 freshly isolated PBMCs were seeded into each well of a 96 well plate, stimulated with 500 U/mL of Interleukin 2 (IL-2; R&D Systems, Minneapolis, USA) and either 1000 (1% responder to stimulator cells) or 100 (0.1% responder to stimulator cells) irradiated OMLP-PCLs were added. Following 3 days of culture. A total of 0.5 µCi/well 3H-thymidine (GE Healthcare, USA) was added to each well for 8 h before the plates were frozen at −20 °C for at least 24 h. After thawing, 3H-labeled cellular material was harvested onto filtermats (PerkinElmer) and activity was counted using a Wallac 1450 MicroBeta-TriLux 3 Detector (PerkinElmer). Data is presented as the mean ± SD from n = 6 technical repeats.
As part of the assay, to prevent the OMLP-PC cell lines from proliferating, they were irradiated with 20 Gy (iOMLP-PCL). 1 × 105 freshly isolated PBMCs were seeded into each well of a 96 well plate, stimulated with 500 U/mL of Interleukin 2 (IL-2; R&D Systems, Minneapolis, USA) and either 1000 (1% responder to stimulator cells) or 100 (0.1% responder to stimulator cells) irradiated OMLP-PCLs were added. Following 3 days of culture. A total of 0.5 µCi/well 3H-thymidine (GE Healthcare, USA) was added to each well for 8 h before the plates were frozen at −20 °C for at least 24 h. After thawing, 3H-labeled cellular material was harvested onto filtermats (PerkinElmer) and activity was counted using a Wallac 1450 MicroBeta-TriLux 3 Detector (PerkinElmer). Data is presented as the mean ± SD from n = 6 technical repeats.
4
Immunosuppressive Potential of hUCB Cells
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The immunosuppressive ability of hUCB cells was examined in a proliferation assay with stimulated peripheral blood mononuclear cells (PBMCs), as previously described [27 (link)]. Briefly, human PBMCs were isolated from adult peripheral blood and seeded in 96-well, flat-bottom microtiter plates (Nunc, Thermo Fisher Scientific, Australia). Assays were performed in triplicate at a concentration of 2.5 × 105 PBMCs per well in complete RPMI-1640 medium alone or in the presence of 800 ng/ml ionomycin and 20 pg/ml PMA (Sigma-Aldrich, Australia) to a final volume of 200 μl per well. For wells that required the addition of hUCB cells, 50 μl of hUCB cells at hUCB: PBMC ratios ranging from 1:1 to 1:40 was added to each well before the addition of PBMCs. Cells were incubated at 37 °C for 48 h, and then, 1 μCi/well [3H]-thymidine (Perkin Elmer, Australia) was added for an additional 18 h of culture. Cells were harvested onto filter mats (Perkin Elmer, Australia), and incorporated radioactive nucleic acids were counted using a Top Count Harvester (Packard Biosciences, Meriden, CT USA).
5
Allogeneic Lymphocyte Proliferation Assay
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Mononuclear cells from lymphoid tissues were prepared as above, and mixed lymphocyte reactions were performed. In brief, cells were seeded in 96-well, flat-bottom microtiter plates in triplicate. Splenocytes (2 × 105) from C57BL/6 mice (effectors) were incubated with equal numbers of irradiated (20 Gy) Balb/c stimulators or hAECs. In experiments involving inhibition of allogeneic proliferation, hAECs were added as third-party cells at a hAEC to splenocyte ratio of 1:5 prior to the addition of splenocytes. Cells were incubated at 37 °C for 4 days and then 1 μCi/well [3H]-thymidine (Perkin Elmer) was added for an additional 18 h of culture. Cells were harvested onto filter mats (Perkin Elmer) and incorporated radioactive nucleic acids counted using a Top Count Harvester (Packard Biosciences).
6
CD8+ T cell Proliferation Assay
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PBMCs were isolated from 50 healthy donor buffy coats and CD8+ T cells were depleted using CD8+ RosetteSep (StemCell Technologies). Cells were plated at a density of 4–6 million cells per mL in AIM-V medium (Gibco). On days 5, 6, and 7, cells were gently resuspended in 3 × 100 μL aliquots and transferred to each well of a round bottomed 96 well plate. Cultures were pulsed with 0.75 μCi [3H]-Thymidine (Perkin Elmer) in 100 μL AIM-V culture medium, and incubated for a further 18 h, before harvesting onto filter mats (Perkin Elmer), using a TomTec Mach III cell harvester. Counts per minute (Cpm) for each well were determined by Meltilex (Perkin Elmer) scintillation counting on a 1450 Microbeta Wallac Trilux Liquid Scintillation Counter (Perkin Elmer) in paralux, with low background counting.
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