BMDCs seeded in 96-well cell culture plates at a density of 1 × 105/ml in 500 μl of complete medium were incubated for 1 h with H3-adenosine at final concentrations of 0–12,000 nM in triplicate, then cell-bound and free H3-adenosine were separated by harvesting the cells on a cell harvester (PerkinElmer) and the cell-associated radioactivity measured by liquid scintillation. Scatchard plot analysis was then performed and the dissociation constant and maximum binding capacity calculated.
Cell harvester
Manufactured by PerkinElmer
Sourced in United States
The Cell Harvester is a versatile laboratory instrument designed for the efficient collection and isolation of cells from cell culture samples. It facilitates the separation of cells from their growth medium, enabling further analysis or processing. The core function of the Cell Harvester is to provide a consistent and reliable method for harvesting cells, ensuring the preservation of cell viability and integrity.
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Lab products found in correlation
Topcount scintillation counter, by PerkinElmer (3 mentions)
Topcount instrument, by PerkinElmer (2 mentions)
Betaplate scint solution, by PerkinElmer (1 mentions)
Wallac 1450 microbeta trilux liquid scintillation counter, by PerkinElmer (1 mentions)
Wallac microbeta scintillation counter, by PerkinElmer (1 mentions)
Vi cell xr cell viability analyser, by Beckman Coulter (1 mentions)
α cd3 cd28 beads, by Thermo Fisher Scientific (1 mentions)
Gamma counter, by PerkinElmer (1 mentions)
3h thymidine, by GE Healthcare (1 mentions)
3h hypoxanthine, by PerkinElmer (1 mentions)
Liquid beta scintillation counter, by Hewlett-Packard (1 mentions)
Microscint 0, by PerkinElmer (1 mentions)
Gf c filter plates, by PerkinElmer (1 mentions)
3h pirenzepine, by GraphPad (1 mentions)
Prism 5, by GraphPad (1 mentions)
Pirenzepine, by GraphPad (1 mentions)
Topcount, by PerkinElmer (1 mentions)
3h pirenzepine, by PerkinElmer (1 mentions)
Microbeta2 microplate scintillation counter, by PerkinElmer (1 mentions)
Microbeta2 microplate counter, by PerkinElmer (1 mentions)
23 protocols using cell harvester
1
Adenosine Binding Kinetics in BMDCs
2
Competitive Binding Assay for Amyloid Ligands
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A fixed concentration of [3H]DCVJ (10 nM) a pan-amyloid ligand (Kd for α-syn 4.4 nM, Aβ 8.9 nM, tau 15.6 nM), and α-syn (125 nM) prepared as described previously (Paslawski et al., 2016 (link), 2019 (link)) were used with different concentrations of d 2, d 4, d 6, and d 8 from 0.01 to 100 nM. The competitor reaction was diluted with 20 mM Tris-HCl, pH 7.4 to a final volume of 200 μL per well. After incubation for 2 h at 37°C, the binding mixtures were filtered through a Perkin Elmer GF/B glass filter via a TOMTEC cell harvester and immediately washed three times with 1 mL of deionized water. Filters containing the bound ligands were dried and mixed with 3 mL of Betaplate scint solution (PerkinElmer Life Sciences) and incubated for 2 h before counting in a Wallac 1450 MicroBeta TriLux Liquid Scintillation Counter (PerkinElmer Life Sciences). For the determination of the inhibition constants, data were analyzed using GraphPad Prism software (version 7.0) to obtain IC50 values by fitting the data to the equation Y = bottom + (top - bottom)/(1 + 10(x - log IC50). Ki values were calculated from IC50 values using the equation Ki = IC50/(1 + [radioligand]/Kd).
3
Evaluating VP-16's Impact on Lymphocyte Cytotoxicity
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To test the influence of VP-16 on cytotoxic activity of lymphocytes we used JAM assay. Effector lymphocytes were firstly stimulated and incubated with VP-16 either for 24 or 72 h and mixed with the target tumor cell line in different ratios. In the first experiment, the effector cells were stimulated in vitro with the soluble anti-CD3/CD28 antibodies and cultured with 0.1 μg/mL VP-16 for 3 days. In the other experiment, VP-16 was washed out after 24 h exposition followed by 48 h stimulation with the antibodies. The target cells were P815 mastocytoma cell line which functions in a cell-mediated cytotoxicity manner. One day before the test, the target cells were incubated with [3H]-thymidine (10 mCi per 1 mL) overnight, then coated with the soluble anti-CD3 antibody for 1 h and washed. The assay was performed on V-shaped 96-well plate. The target cells were co-cultured with the stimulated lymphocytes at a particular effector/target ratio (25:1, 12.5:1, 6.25:1, 3:1, 1.5:1, 0.75:1) for 6 h. The lymphocytes were subsequently harvested (using Perkin Elmer Cell Harvester) and the radioactivity was measured using a Wallac Micro Beta scintillation counter (Perkin Elmer).
4
Woodchuck PBMC Proliferation Assay
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Antigen-specific proliferation of woodchuck PBMCs was determined by 2[3H]-adenine-based assay as described previously [49] (link). Briefly, 5×104 PBMCs were stimulated with 5 µg/ml purified WHcAg protein for 5 days. Unstimulated cells served as a negative control. Afterwards, cells were labelled with 1 µCi of 2[3H]-adenine (Hartmann Analytic, Braunschweig, Germany) for 16 h and collected using a cell harvester (Perkin Elmer, Waltham, MA). Results for triplicate cultures are presented as a mean stimulation index (SI). SI is calculated with the formula: (stimulated cpm - blank cpm)/(unstimulated cpm - blank cpm).
5
Measuring A2BR Adenosine Receptor Binding
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BMCs or in vitro cultured BMDCs were seeded in 96‐well cell culture plates at a density of 1×105/mL in 100 μL of complete medium were incubated at 37°C for 1 h with H3‐adenosine at final concentrations of 1.0 μM in triplicate, with or without selective A2BR antagonist MRS 1754 (1 μM), then cell‐bound and free H3‐adenosine were separated by harvesting the cells on a cell harvester (Perkin Elmer) and the cell‐associated radioactivity measured by liquid scintillation.
6
Allogeneic Mixed Lymphocyte Reaction
7
Murine and Human T Cell Proliferation Assay
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Murine splenic T cells were collected as described above and plated out in triplicate in a 96-well plate containing 100 000 cells per well. Cells were stimulated with either PMA [50 ng/ml] combined with ionomycin [1000 ng/ml] or with αCD3/CD28 beads [Thermofisher Scientific, 10 splenocytes/bead] in presence or absence of TYK2i [10‐10 000 nM]. After 2 days, tritium [3H; Sigma] was added [0.3 μl/50 µl]. After a further 24 h, cells were harvested using the Cell Harvester [Perkin Elmer, Waltham, MA, USA]. In human T cell activity assays, lymphocyte proliferation was induced either by adding αCD3/CD28 beads [10 lymphocytes/bead] or by mixing peripheral blood mononuclear cells [PBMCs] in a 1 to 1 ratio [mixed lymphocyte reaction], in presence or absence of TYK2i [10‐10 000 nM]. To determine human lymphocyte viability in presence of TYK2i, lymphocytes were treated with TYK2i [2000 nM] or DMSO for 24 h . Next, cell viability was verified using Vi-CELL XR Cell Viability Analyser [Beckman Coulter], based on trypan blue dye exclusion method in accordance with the manufacturer’s instructions. The viability was reported as percentage live cells.
8
Biparatopic Nanobodies Inhibit Breast Cancer Cell Proliferation
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Example 15
The growth inhibitory characteristics of biparatopic Nanobodies were evaluated using the breast tumor cell line SKBR3. Briefly, SKBR3 cells were detached using 0.25% (vol/vol) trypsin and suspended in DMEM supplemented with 10% fetal calf serum (FCS), glutamine, and penicillin-streptomycin at a density of 1×105 cells/ml. Aliquots of 200 μl (2×104 cells) were plated into 96-well microdilution plates and allowed to adhere. After overnight adherence, cells were washed with serum-free medium and starved for 4 hours in 100 μl serum-free medium. Then, 100 μl of 1% FCS containing medium alone or 90 μl of 1% FCS containing medium with serial dilutions of IMAC/SEC purified biparatopic Nanobodies, monovalent 2D3 or 50 nM Herceptin® was added. After 2 days of incubation, cells were pulsed with 1 μCi [3H]-thymidine and incubated for an additional 24 h prior to freezing at −80° C. Cells were subsequently thawed and embedded on glass fiber membranes using a cell harvester (Perkin Elmer Life Sciences, Wellesley, Mass., USA). After several washings with water, filters were air-dried and counted using a γ-counter (Perkin Elmer Life Sciences). Biparatopic Nanobodies are able to inhibit cell proliferation of SKBR3 cells to an equal or greater extent than Herceptin®.
9
Thymidine Incorporation for Cell Proliferation
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The measure of cell proliferation was assessed by titrated thymidine incorporation, as described previously 24 , 25 . Briefly, human fibroblasts were seeded in quadruplicates in a 96‐well plate in complete medium (4000 cells/well). Cells were starved in serum‐free MEM for 24 h. FBS (10%) or PDGF‐BB (25 ng/mL) was then added with [3H]‐thymidine (0.5 μCi/well; GE Healthcare) for 24 h. Some experiments were also performed in the presence of palmitate, palmitoleate, stearate, oleate (all at 0.2 mm ), SCD inhibitor (2–10–20 μm ), or vehicle. Microtiter plates were harvested using a cell harvester (PerkinElmer Life Sciences, Zaventem, Belgium). The radioactivity incorporated into DNA was quantified using a TopCount instrument (PerkinElmer Life Sciences).
10
Modulating Malaria Parasite Growth
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Ring-stage erythrocytes (1.5% parasitemia) were treated with MβCD for 30 min and washed 3 times with normal medium. The hematocrit was maintained at 2.5%. MβCD-treated ring-stage erythrocytes were then treated with either MβCD-cholesterol, MβCD-desmosterol, or MβCD-epicholesterol for 30 min and washed 3 times with low-hypoxanthine medium. The hematocrit was then adjusted to 1.5% by addition of low-hypoxanthine medium to the cells. We then plated 200 μL of cells in triplicates in a 96-well plate. Each well was pulsed with 22 μL of 0.5 μCi/mL [3H]-hypoxanthine (PerkinElmer; product no. NET177) and incubated for 24 h at 37°C in a 5% CO2, 5% O2, and 90% N2 chamber. Parasites were lysed by freeze-thaw and were collected on filters using a cell harvester (PerkinElmer Life Sciences). This was followed by addition of MicroScint-O scintillation cocktail, and incorporation of [3H]-hypoxanthine was measured using a TopCount scintillation counter (PerkinElmer Life Sciences).
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