Incorporation of 3[H]-hypoxanthine was performed as previously described [21 (link)] with some modifications. Briefly, a vial of the P. falciparum NF54 and 7G8 cell banks was thawed, as outlined above, and immediately added in quadruplicate to a 96-well flat bottom plate at 2% haematocrit in RPMI-1640 (Gibco) supplemented with 10% heat inactivated human serum and 0.01 mg/ml gentamicin (Gibco). Non-parasitized human erythrocytes were added to the plate as a control. Parasitized and non-parasitized erythrocytes were incubated in the presence of 0.5 μCi/well of 3[H]-hypoxanthine (Amersham Pharmacia) for up to 36 hours. Plates were then placed at −80°C overnight. Following thawing, cells were harvested onto glass fibre mats (Perkin Elmer) using a Filtermate cell harvester (Perkin Elmer) and 3[H]-hypoxanthine incorporation was determined using a Microbeta2 Microplate Counter (Perkin Elmer).
Filtermate cell harvester
Manufactured by PerkinElmer
Sourced in Australia, United States
The Filtermate cell harvester is a laboratory equipment used for the rapid and efficient collection of cells from multi-well plates. It operates by filtering cell suspensions through a membrane, capturing the cells while allowing the liquid to pass through. The device is designed to automate the cell harvesting process, improving consistency and throughput compared to manual methods.
Automatically generated - may contain errors
Lab products found in correlation
Glass fibre mats, by PerkinElmer (3 mentions)
Topcount nxt microplate scintillation counter, by PerkinElmer (3 mentions)
Microscint 20, by PerkinElmer (2 mentions)
Gentamicin, by Thermo Fisher Scientific (1 mentions)
3h hypoxanthine, by Cytiva (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Microbeta2 microplate counter, by PerkinElmer (1 mentions)
β scintillation microplate counter, by PerkinElmer (1 mentions)
3h thymidine, by PerkinElmer (1 mentions)
Microbeta2 counter, by PerkinElmer (1 mentions)
Prism 5, by GraphPad (1 mentions)
Human serum, by LGC (1 mentions)
Topcount nxt scintillation counter, by PerkinElmer (1 mentions)
X vivo 15, by Lonza (1 mentions)
Interleukin 2 (il 2), by R&D Systems (1 mentions)
Unifilter gf b plates, by PerkinElmer (1 mentions)
Microbeta liquid scintillation counter, by PerkinElmer (1 mentions)
Easysep negative human cd4 kit, by STEMCELL (1 mentions)
Methyl 3h thymidine, by Cytiva (1 mentions)
Topcount, by PerkinElmer (1 mentions)
18 protocols using filtermate cell harvester
1
Quantifying Plasmodium falciparum Proliferation
2
PBMC Proliferation Assay with Tritium
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For assessing proliferation of PBMCs via the incorporation of radioisotope, the unlabelled cells were pulsed with 1 μCi of 3[H]-thymidine/well (Perkin Elmer, Australia) for the final 18 h and plates were stored at − 80 °C. Following thawing, cells were harvested onto glass fibre mats (Perkin Elmer, Australia) using a Filtermate cell harvester (Perkin Elmer) and radioactivity was measured using a β-scintillation microplate counter (Perkin Elmer). The uptake of 3[H]-thymidine was measured as corrected counts per minute (CCPM), and results were expressed as deltaCPM, which is defined as the 3[H]-thymidine (CPM) in the presence of stimulus, subtracting the average 3[H]-thymidine (CPM) incorporated in the presence of the appropriate control stimulus (e.g. unparasitised red blood cells).
3
Assessing Parasite Viability after Chemical Attenuation
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The viability of the parasites following chemical attenuation was assessed using the [3H]-hypoxanthine growth inhibition assay. Chemically attenuated ring-stage parasites (2% haematocrit) were added to 96-well flat-bottomed plates (100 μl per well) in quadruplicate. Unattenuated ring-stage parasites and unparasitised red blood cells (uRBC) at 2% haematocrit were used as positive and background controls respectively. Plates were placed in a 37 °C incubator with 5% O2, 5% CO2, and 90% N2. The assay duration was 48 h with [3H]-hypoxanthine (0.2 μCi/well) added from the start of the experiment. Following incubation, plates were frozen, then subsequently thawed and harvested onto glass fibre mats (Perkin Elmer, Australia) using a Filtermate cell harvester (Perkin Elmer). Radioactivity was measured using a Microbeta2 counter (Perkin Elmer). The remainder of the packed cells from the vaccine were placed in culture, and after 1 week, 2 weeks and 3 weeks of culture, cells were harvested and evaluated according to incorporation of [3H]-hypoxanthine. Twice a week, fresh uRBC were added into the cultures and the medium changed. No growth was observed, as measured by lack of [3H]-hypoxanthine incorporation, compared to unattenuated Pf 7G8 control samples that were cultured in parallel.
4
Radioligand Binding Assay Protocol
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Inhibition radioligand
binding assays were conducted as previously described48 (link),49 (link) with 0.2 nM [3H]NMS in binding buffer in a final volume
of 250 μL. Nonspecific binding was defined in the presence of
10 μM atropine. Briefly, membrane fractions (about 25–70
μg/mL of protein) were incubated with radioligand and unlabeled
test compounds for 2 h at r.t. Bound and free radioactivity were separated
by filtering the assay mixture through UniFilter GF/B plates using
a FilterMate Cell Harvester (PerkinElmer Life and Analytical Science).
The filter bound radioactivity was counted by a TopCount NXT Microplate
Scintillation Counter (PerkinElmer Life and Analytical Science). Data
(cpm) were normalized to percentage-specific binding and analyzed
using a four-parameter logistic equation in GraphPad Prism 5.02; IC50 values were determined, and Ki values were calculated.59 (link) The values reported inTables 1 and 2 represent the
arithmetic mean ± S.E.M. of at least three independent experiments,
each one performed in duplicate.
binding assays were conducted as previously described48 (link),49 (link) with 0.2 nM [3H]NMS in binding buffer in a final volume
of 250 μL. Nonspecific binding was defined in the presence of
10 μM atropine. Briefly, membrane fractions (about 25–70
μg/mL of protein) were incubated with radioligand and unlabeled
test compounds for 2 h at r.t. Bound and free radioactivity were separated
by filtering the assay mixture through UniFilter GF/B plates using
a FilterMate Cell Harvester (PerkinElmer Life and Analytical Science).
The filter bound radioactivity was counted by a TopCount NXT Microplate
Scintillation Counter (PerkinElmer Life and Analytical Science). Data
(cpm) were normalized to percentage-specific binding and analyzed
using a four-parameter logistic equation in GraphPad Prism 5.02; IC50 values were determined, and Ki values were calculated.59 (link) The values reported in
arithmetic mean ± S.E.M. of at least three independent experiments,
each one performed in duplicate.
5
CD4+ T Cell Proliferation Assay
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[3H]thymidine incorporation assay was performed as previously described [44 (link)]. Briefly, CD4+CD25- T cells were suspended in X-VIVO-15 (Lonza) media supplemented with 10% human serum (SeraCare, Milford, MA) and penicillin/streptomycin, then seeded into a 96 well flat bottom plate in a ratio of 10:1 with loaded or unloaded autologous (self) DCs. Wells were supplemented with 10 units/ml of IL-2 (R&D Systems) on day 0 and cultured at 37°C for six days. On day 6, unless otherwise indicated, wells were pulsed with radioactive thymidine (PerkinElmer Waltham, MA) for 6 hours then harvested using a Filtermate cell harvester (PerkinElmer,). Thymidine incorporation was quantified using a TopCount NXT scintillation counter (PerkinElmer). To calculate the stimulation index 1×105 CD4+CD25- T cells were stimulated with 1×104 autologous DCs loaded with survivin (DC:survivin) in a 96 well flat bottom tissue culture plate for 6 days. Proliferation for each well was determined as described above. The stimulation index for each well was calculated against T cells similarly stimulated using unloaded autologous DCs (> = 10 replicates per donor). Stimulation Index = [1×105 CD4+CD25- T cells stimulated with 1×104 DC:survivin (numerator)]/[Mean of > =10 DC:null stimulated T cell controls (denominator)].
6
Radioligand Binding Assay for Muscarinic Receptors
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The inhibition binding experiments were carried out at room temperature in polypropylene 96-well plates (Sarstedt, Verona, Italy) in a final volume of 250 μL of 25 mM Na/K phosphate buffer containing 5 mM MgCl2 at pH 7.4 (assay buffer), in the presence of 0.2 nM [3H]-N-methylscopolamine chloride ([3H]-NMS) (PerkinElmer Life and Analytical Science) and different concentrations of the tested compounds (0.001–100 µM). Aliquots of membranes (25–70 µg/mL) were added and incubated for 2 h at room temperature, filtered through UniFilter GF/B plates (PerkinElmer Life and Analytical Science, Monza, Italy) using a FilterMate cell harvester (PerkinElmer Life and Analytical Science, Monza, Italy). The filters were washed several times with ice-cold MilliQ water. Then, the plates were counted in a β-counter (TopCount NXT microplate scintillation counter, PerkinElmer Life and Analytical Science, Monza, Italy).
7
In Vitro Lymphocyte Proliferation Assay
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Blood samples drawn on the same day, were simultaneously sent to the Central Laboratory of the University Hospitals Leuven, Belgium, where LPT was performed according to the protocol described in the ATS official statement on diagnosis and management of beryllium sensitivity and chronic beryllium disease [32 (link)].
On day 7, 50 μl (1:50 diluted) of [3H] thymidine was added per well. Cells were collected 6 h later on glass-fiber filters using a cell harvester (PerkinElmer Filtermate Cell Harvester). The incorporated radioactivity was measured using a β-counter (PerkinElmer Tricarb 2910 TR Liquid Scintillator Analyzer), to determine extent of cell proliferation. Lymphocyte proliferation results are expressed as Stimulation Index (SI) which is calculated by dividing the mean counts/min (cpm) in stimulated conditions by the mean cpm in unstimulated control wells. [3H] thymidine LPT derived SI (LPT-SI) of >2.5 was considered as an abnormal or positive response [33 (link)].
On day 7, 50 μl (1:50 diluted) of [3H] thymidine was added per well. Cells were collected 6 h later on glass-fiber filters using a cell harvester (PerkinElmer Filtermate Cell Harvester). The incorporated radioactivity was measured using a β-counter (PerkinElmer Tricarb 2910 TR Liquid Scintillator Analyzer), to determine extent of cell proliferation. Lymphocyte proliferation results are expressed as Stimulation Index (SI) which is calculated by dividing the mean counts/min (cpm) in stimulated conditions by the mean cpm in unstimulated control wells. [3H] thymidine LPT derived SI (LPT-SI) of >2.5 was considered as an abnormal or positive response [33 (link)].
8
Inactivated CHIKV Vaccine-Induced Splenocyte Proliferation
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Mice were vaccinated s.c. with 10 µg of inactivated CHIKV as described [48] (link). Standard proliferation assays using tritiated thymidine uptake were undertaken using splenocytes isolated 3 weeks post vaccination. Briefly, splenocytes (2.5×105 cells/96 well, 6 replicates) were cultured with 10 µg/ml of inactivated CHIKV [48] (link) for 3 days, tritiated thymidine was then added and cells harvested the next day onto a MicroBeta Filtermat-96 A using the FilterMate™ Cell Harvester (PerkinElmer). Radioactivity was measured using the MicroBeta Liquid Scintillation Counter (PerkinElmer).
9
Antigen-Loaded moDC Stimulate T-Cell Proliferation
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The moDCs were obtained as described above and loaded with protein antigen (0.3 μM final concentration, for Nbs, mAbs, and KLH) for 5 h. Cells were washed three times with unsupplemented RPMI culture medium, followed by resuspension in DC medium supplemented with or without maturation cocktail (1,000 IU/ml TNF-α, 10 ng/ml IL-6, 10 ng/ml IL-1β, and 5mM PGE2) and left overnight at 37°C. Cells were washed again three times with unsupplemented RPMI medium and resuspended in DC medium. The next day, T-cells were isolated from PBMCs with the EasySep Negative Human CD4 kit (Stemcell) according to the manufacturer's instructions and T-cells were co-cultured with autologous moDCs at a 10:1 ratio in presence of 3H-thymidine. After 6 days, T cell proliferation during the last 15 h of co-culture was estimated by measuring the counts per minute (cpm) of incorporated 3H-thymidine in harvested cells (FilterMate Cell Harvester, Perkin Elmer) with a β-counter. The stimulation index (SI) was calculated as the ratio between counts per minute (cpm) obtained in cultures with moDCs loaded with protein antigen plus autologous T-cells and cpm obtained in cultures containing moDCs, which were not loaded plus autologous T-cells.
10
Allogeneic Lymphocyte Proliferation Assay
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Allogeneic peripheral blood mononuclear cells were seeded in RPMI 1640 and 10% FBS and monocytes were let to adhere. Leukocytes remained in suspension were collected, centrifuged at 1200 rpm for 10 min and used for MLR, as previously published [19] (link). Briefly, lymphocytes were seeded into 96-well plates at 105 cells/well and purified allogeneic DCs (5×103) were added to each well and incubated for 5 days at 37°C in a humidified chamber in air and 5% CO2. For the last 18 h, 1 µCi of [methyl-3H]thymidine (Amersham Pharmacia Biotech, Little Chalfont, UK) was added to each well. Cells were finally collected by a Filtermate Cell Harvester (Perkin–Elmer, Waltham, MA, USA). 3H-Thymidine uptake was quantified by liquid scintillation counting on a TopCount NXT Microplate Scintillation Counter (Perkin–Elmer). Each experiment was performed in triplicate.
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