Splenocytes from 4C mice (C57BL/6 transgenic for a TCR recognizing the BALB/c MHC I-Ad) (23 (link)) were used as responders against irradiated BALB/c splenocytes as stimulators in a standard MLR assay (19 (link)). Briefly, 4C splenocytes were cultured in a Petri dish for 45 minutes at 37°C to enrich for T cells. Non-adherent cells were collected, washed, and incubated with 2000 cGy irradiated BALB/c splenocytes in 96-well U-bottom plates (105 cells/well/each) in complete MLR medium (19 (link)). After 48 hrs of incubation, cultures were supplemented with varying concentrations of SA-PDL1 protein or equimolar concentrations of control SA protein. Cultures were incubated for a total of 72 hrs, with the last 16 hrs pulsed with [3H] thymidine (1 μCi/well). Cultures were then harvested with Tomtec cell harvester to assess DNA-associated radioactivity [counts per minute (cpm)] as the measure of cell proliferation using a Beta plate counter. The percentage inhibition of T cell proliferation was calculated using the formula of 1- [cpm in test proliferation / cpm in control proliferation] x 100.
Cell harvester
Manufactured by Tomtec
Sourced in United States
The Cell Harvester is a piece of laboratory equipment used to collect and isolate cells from a solution. It functions by filtering the solution through a membrane, capturing the cells while allowing the surrounding liquid to pass through. The core purpose of the Cell Harvester is to efficiently separate and collect cellular material for further analysis or experimentation.
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Lab products found in correlation
3h thymidine, by Cytiva (2 mentions)
Cd4 microbeads, by Miltenyi Biotec (1 mentions)
Ls column, by Miltenyi Biotec (1 mentions)
Cd11c microbeads, by Miltenyi Biotec (1 mentions)
Mitomycin c, by Merck Group (1 mentions)
Multitest 6 color tbnk reagent, by BD (1 mentions)
Novocyte, by Agilent Technologies (1 mentions)
Facs lysing solution, by BD (1 mentions)
Interleukin 2 (il 2), by R&D Systems (1 mentions)
Microbeta, by PerkinElmer (1 mentions)
Microbeta plate counter, by PerkinElmer (1 mentions)
3h 8 oh dpat, by PerkinElmer (1 mentions)
Betaplate 1205 liquid scintillation counter, by PerkinElmer (1 mentions)
Glass fibre filter, by PerkinElmer (1 mentions)
15 protocols using cell harvester
1
Measuring T Cell Proliferation Inhibition
2
Saturation Binding of MOP-r Agonist DAMGO
3
Isolation and Characterization of Dendritic Cells
4
Evaluating Tolerogenic Dendritic Cell Immunomodulation
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Tolerogenic DCs were generated as described above (“Differentiation of primary human DCs and generation of knockout DCs”) and resuspended to a density of 4 × 105 cells/mL. TGFβ-treated (2.5 ng/mL, overnight) wild-type or fibronectin knockout LX-2 cells were treated with 25 μg/mL mitomycin C (Sigma-Aldrich, catalog no. M7949) to inhibit cell proliferation for 1 hour at 37°C, washed, and resuspended to a density of 1 × 105 cells/mL. Allogeneic T cells were purified from PBMCs by negative selection using the Miltenyi human pan T Cell Isolation Kit (Miltenyi Biotec, catalog no. 130-096-535) and resuspended to a density of 2 × 106 cells/mL. Wells of a 96-well, round-bottom plate were prefilled with 50 μL of anti-KLH or anti-ILT3 antibody at 20 μg/mL. Mixed lymphocyte reactions were established by adding 50 μL of each cell type at the densities indicated above, for a final T cell:DC:LX-2 cell ratio of 20:2:1. After 5 days, the media was replaced with fresh media containing tritiated thymidine (3H-thymidine; PerkinElmer, catalog no. NET027E001MC) at a concentration of 1 μCi/mL. After an additional 18 hours of incubation, cells were harvested onto filters using a cell harvester (Tomtec) and incorporation of radiolabeled thymidine was counted on a MicroBeta2 microplate reader (PerkinElmer).
5
Suppression Assay for Regulatory T-Cell Function
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Example 8
Suppression assays were performed in round bottom 96-well microtiter plates. 100,000 responder PBMC from same cell source as sorted populations, 30,000 sorted cells (one of 7 different sorted subtypes based on CD25 and/or CD127 expression), 100,000 allogeneic irradiated CD3-depleted PBMC were added as indicated. Responder ratio indicated refers to Treg to responder where 1 sorted: 1 responder is 30,000 sorted cells: 100,000 PBMC responder cells. APC consisted of allogeneic PBMC depleted of T cells using StemSep human CD3+ T cell depletion per manufacturer's recommendations (StemCell Technologies, Seattle, Wash.) and irradiated with 40 Gy. Cells were plated in the following order in 50 ul per well: sorted cells, responders, APC. No additional stimulus was added to the wells, however, additional media was added to each well so the final volume was 200 ul per well. Wells surrounding culture wells were filled with PBS in order to prevent evaporation. T cells were incubated for 7 days at 37° C. in 5% CO2. Sixteen hours before the end of the incubation, 1 uCi 3H-thymidine was added to each well. Plates were harvested using a Tomtec cell harvester and 3H-thymidine incorporation was determined using a 1450 microbeta Wallac Trilux liquid scintillation counter.
6
Lymphocyte Blast Formation and Proliferation
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Blast formation was studied by flow-cytometric assay for specific cell-mediated immune-responses in activated whole blood (FASCIA). Heparinized blood was diluted in RPMI medium containing Glutamax I, gentamicin, and β-mercaptoethanol and stimulated with IL-2 (R&D Systems) in concentrations of 40 U/ml and 320 U/ml for 5 days. After stimulation, cells were stained with Multitest 6-Color TBNK reagent (BD) including CD3, CD45, CD4, CD8, CD19, and CD16/CD56 antibodies. After red blood cells were lysed with FACS lysing solution (BD), lymphocyte subpopulations were analyzed for the percentage of blasts based on their light scatter characteristics using flow cytometry (NovoCyte; Acea). In studying lymphocyte proliferation, purified PBMCs were incubated in culture medium (2 × 105 cells per well) in 96-well polystyrene plates in the presence of increasing concentrations of IL-2 (R&D Systems) for 72 h. For the last 6 h, 1 μCi of 3H-Thymidine was added to each well. All conditions were performed in triplicates. Cells were harvested using a cell harvester (Tomtec) and thymidine incorporation was measured with beta-scintillation counter (MicroBeta; Perkin Elmer).
7
Lymphocyte Proliferation Assay
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PBMCs (2×105/well) were seeded in 96-well microtiter plates in 200 µl of culture medium and incubated at 37°C with: i) medium alone (negative control); ii) PHA (2 µg/ml) (positive control), or iii) a pool of all Tat peptides (2 µg/ml of each peptide), in triplicate wells. After 5 days, cells were pulsed with 1 µCi/well of [3H] thymidine (Amersham, Buckinshamshire, United Kingdom) and harvested 18 hours later onto filter paper using a cell harvester (Tomtec, Orange, CT, USA). The incorporated radioactivity was measured in a β-counter. The stimulation index (S.I.) was determined as the ratio between the mean counts/minute of antigen-stimulated wells and the mean counts/minute of the same cellular sample incubated with medium alone (negative control). Stimulation indices (S.I.) ≥3 were considered positive.
8
IL-6 Bioassay in Avian Plasma
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IL6 activity in chicken and duck plasma was measured by the ability to stimulate the proliferation of 7TD1 murine hybridoma cells in a 3H-thymidine incorporation assay. Briefly, 7TD1 cells were cultured in DMEM supplemented with 5% FBS, glutamine, 0.55 mM arganine, 0.24 mM asparagine, penicillin (100 U)/streptomycin (100 μg/mL), and recombinant ChIL6 (100 ng/mL) until the cells reached the logarithmic phase of growth. The cells were washed three times with DMEM to remove the residual ChIL6 in the medium and cultured further for 3 days in the medium without ChIL6. Test samples were serially diluted in 96-well Nunc tissue culture plates, then starved 7TD1 cells were added to each well, and plates were incubated at 37 °C in a humidified CO2 incubator for 48 h. For the last 5 h of incubation, 0.5 Ci of 3H-thymidine (Amersham Biosciences, Buckinghamshire, UK) was added to each well, and cells were harvested onto glass fiber filter mats using a cell harvester (Tomtec, Hamden, CT). Filters were placed in plastic pouches with 5 mL scintillant, and radioactivity was measured on a MicroBeta TriLux 1450 β counter (EG&G Wallace, Turku, Finland). The presence of the IL6 in chicken and duck sera was further confirmed by inhibition of its activity in the 7TD1 bioassay by the addition of rabbit anti-ChIL6 anti-sera [61]. Preimmunization sera were used as a negative control serum.
9
Cell Proliferation Assay for Compound Screening
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The cells were seeded in round-bottom 96-well plates (10,000/well) in the presence of serial dilutions of compounds in DMSO (0.5% final DMSO concentration) and incubated for 72 hours. During the last 8 hours of incubation, the cells were pulsed with [Methyl-3H]-Thymidine and then harvested onto glass fiber filtermats using a Tomtec Cell Harvester. Filters were counted with a Wallac Microbeta 1405 Scintillation Beta Counter. Radioactivity associated to each sample is proportional to the amount of labelled thymidine incorporated into newly synthesized DNA, giving a direct measure of the cell proliferation rate. All values are normalized to vehicle-treated control which is set as 100% proliferation. The Inhibitory Concentration 50 (IC50) value is defined as the inhibitor concentration that yields 50% proliferation, relative to control. IC90 represents the concentration that causes 90% inhibition.
10
Radioligand Binding Assay for nAChR
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