Peripheral blood mononuclear cells (PBMC) were separated from fresh heparinized whole blood obtained from study participants by density gradient centrifugation. Briefly, 15–30 ml of whole blood was carefully layered onto the membrane a Leucosep tube (Greiner Bio-One) pre-loaded with LymphoPrep™ (StemCell Technologies, France) and centrifuged. PBMC were collected from the interface and washed twice with pre-warmed R0 media (RPMI 1640, 2mM L-glutamine, 100U/ml penicillin and streptomycin and 1% sodium pyruvate; Sigma Aldrich, UK), then resuspended in R10 media (R0 media with 10% fetal calf serum (FCS); Sigma Aldrich) and counted using a CASY® counter (Roche, Switzerland). For cryopreservation, cells were centrifuged and resuspended in ice-cold FCS to which an equal volume of 20% DMSO in FCS was added to give a concentration of 5–10 × 106 cells/ml, aliquoted into cryovials and placed in a CoolCell® cell freezing container which was stored at −80 °C until transfer to liquid nitrogen after 1–2 days. Cryopreserved PBMC were rapidly thawed in a 37 °C waterbath, transferred to a tube containing pre-warmed R10, centrifuged and resuspended in R10 containing 2 μl/ml of benzonase (Merck Chemicals) and rested at 37 °C, 5% CO2, for a minimum of 2 hours before counting and use in subsequent assays.
Casy counter
Manufactured by Roche
Sourced in Germany
The CASY counter is a laboratory instrument used for accurately measuring and analyzing cell samples. It utilizes the Coulter principle to provide precise cell counts and size distributions. The CASY counter is designed to deliver reliable and consistent results for a wide range of cell types.
Automatically generated - may contain errors
Lab products found in correlation
Benzonase, by Merck Group (3 mentions)
Glutamine, by Thermo Fisher Scientific (2 mentions)
Penicillin and streptomycin, by Merck Group (1 mentions)
Leucosep tube, by Greiner (1 mentions)
Rpmi 1640, by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
Lymphoprep, by STEMCELL (1 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Rpmi medium, by Thermo Fisher Scientific (1 mentions)
Victor nivo plate reader, by PerkinElmer (1 mentions)
Methanol, by Merck Group (1 mentions)
10 protocols using casy counter
1
Isolation and Cryopreservation of PBMCs
2
Cryopreserved PBMC Thawing and Resting
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Two vials of cryopreserved PBMC from each sample were rapidly thawed in a 37°C water bath and transferred to a 15-ml Falcon tube containing 10 ml R10 (RPMI medium, 10% fetal calf serum, 1% l -glutamine, 1% penicillin-streptomycin, 1% sodium pyruvate). PBMC were pelleted, supernatants were discarded, and the samples were resuspended in 10 ml R10 with 20 μl Benzonase (25 U/μl) (Merck Chemicals Ltd.) and rested overnight at 37°C and 5% CO2. For the 2-h versus overnight comparison, PBMC were split into equal volumes; one set was rested for 2 h, and one set was rested overnight. PBMC were counted on a Casy counter (Roche) and split into the appropriate volumes for each assay. In some cases, there were inadequate numbers of cells to perform all assays and use all conditions.
3
Rapid Thawing and Overnight Rest for Cryopreserved PBMCs
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Cryopreserved PBMC were rapidly thawed in a 37°C waterbath and transferred to a 15 mL Falcon tube containing 10 mL R10 (RPMI, 10% FCS, 1% L-glutamine, 1% Pen-Strep and 1% sodium pyruvate). PBMC were pelleted, supernatants discarded and resuspended in 10 mL R10 with 20 μL Benzonase (Merck Chemicals Ltd.) and rested overnight at 37°C, 5% CO2. PBMC were counted on a Casy Counter (Roche) and split into appropriate volumes for each assay. Not all assays were performed on all samples.
4
Cryopreservation and Thawing of PBMCs
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PBMC were isolated from heparinized peripheral blood samples using density centrifugation. Cells were counted, resuspended in fetal bovine serum (FBS) and incubated at 4°C for 20 min. An equal volume of FBS containing 20% dimethylsulfoxide (DMSO) was then added and cells aliquoted at a concentration of 5–10 × 106 cells per cryovial, frozen overnight at −80°C and transferred to LN2 the following day. Cryopreserved PBMC were thawed in a water bath at 37°C until a small amount of frozen material remained. Samples were gradually added to 10 ml RPMI (containing 10% FBS and 2 mM l-glutamine) and centrifuged at 1,500 rpm for 7 min. Supernatants were poured off and cells resuspended at an approximate concentration of 2–3 × 106 cells per ml of RPMI (containing 10% fetal calf serum and 2 mM l-glutamine), and 2 μl/ml of 25 U benzonase added to each tube. Cells were rested at 37°C for 2 h with 5% CO2 before counting using a CASY Counter (Roche).
5
Characterization of K-RAS mutant cell lines
6
Blue Light Regulates SEAP Production in CHO-K1 Cells
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To measure the difference in the protein production of CHO-K1 cells, a SEAP assay was conducted. The cells were seeded in a black 96-well plate with 0.3 × 105 cells/mL and transfected using Lipofectamine 3000 (Invitrogen GmbH, Darmstadt, Germany) in accordance with the manufacturer’s protocol with the corresponding plasmids, including the reporter plasmid for SEAP expression driven by the constitutive EF1α promoter. CHO-K1 cells were kept in the dark for 24 h, before being illuminated under varying blue light conditions for 48 h with 450 nm blue-light LEDs. After illumination, the supernatant was transferred to a 96-well plate and heated at 65 °C for 1 h. The remaining cells were washed with 1× PBS, detached, and counted using a CASY counter (Roche Diagnostics Deutschland GmbH, Mannheim, Germany). The supernatant was then spun down at 1250× g for 1 min and mixed with 2× SEAP (20 mM homoarginine, 1 mM MgCl2, and 21% diethanolamine; pH 9.8) buffer before the absorbance was measured at 405 nm after the addition of 120 mM pNPP at intervals of 1 min for 1 h. SEAP activity was calculated as described in [35 (link)].
7
Cell Growth Analysis via Cell Counting and MTT Assay
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We assessed cell growth using two assays: (i) cell counting (shown as main results) and (ii) MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. For cell counting, cells were seeded in 24-well plates in complete medium and allowed to grow for 24h, followed by two washes with phosphate-buffered saline and starvation in serum-reduced medium containing 0.1% FCS for 24h. Stimulation with the ligands (concentrations: 0, 0.001, 0.01, 0.1, 1, 10 and 100nM) was in media containing 0.1% FCS for 5 days. At the end of the experiment, the cells were trypsinized and counted using a CASY counter (Roche Applied Science, UK).
For the MTT assay, cells were seeded in 96-well plates in medium containing 0.1% FCS and 0.5% premium grade bovine serum albumin (free of insulin-like growth factor contaminants (35 (link))), and ligands added after 24h (concentrations: 0, 0.002, 0.01, 0.1, 0.2, 0.625, 1.5, 2.5, 5, 10, 20, 50 and 200nM). The experiment lasted for 5 days and ligands replaced every 48h. After 96h of incubation, MTT reagent (10µl) was added to each well and incubated for a further 24h. Insoluble formazan that was formed at the bottom of the wells was solubilized and absorbance was read at 560nm.
For the MTT assay, cells were seeded in 96-well plates in medium containing 0.1% FCS and 0.5% premium grade bovine serum albumin (free of insulin-like growth factor contaminants (35 (link))), and ligands added after 24h (concentrations: 0, 0.002, 0.01, 0.1, 0.2, 0.625, 1.5, 2.5, 5, 10, 20, 50 and 200nM). The experiment lasted for 5 days and ligands replaced every 48h. After 96h of incubation, MTT reagent (10µl) was added to each well and incubated for a further 24h. Insoluble formazan that was formed at the bottom of the wells was solubilized and absorbance was read at 560nm.
8
Culturing and Aging Human Aortic Smooth Muscle Cells
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Primary HAoSMCs (C-12533; Lot 399Z005) were obtained from PromoCell (Heidelberg, Germany) and cultured in C-22062 growth medium (PromoCell) in accordance with the recommendations of the distributor. The cells were grown in 75-cm2 collagen I-coated culture flasks (VWR, Dietikon, Switzerland) at 37 ℃ in an atmosphere with 5% CO2 until 75–80% confluence. Cells in suspension were counted with a CASY® counter (Roche Diagnostics International Ltd., Mannheim, Germany). HAoSMCs cultured and frozen at passages 3 and 4 were considered as young cells. The HAoSMCs were aged in vitro by replicative senescence (Nakano-Kurimoto et al. 2009 (link); Bielak-Zmijewska et al. 2014 (link)). Thus, HAoSMCs grown and frozen in liquid nitrogen at passages 12 and 13, at which point proliferation had decreased, were considered as old cells. For all experiments, young and old cells were seeded in the same plate before exposure.
9
K-RAS Mutant Lung Cancer Cell Culture
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The human K‐RAS‐mutated lung AC cell lines A549 and A427 were grown in RPMI medium (Gibco, Grand Island, NY) supplemented with 10% FBS, 2 mM glutamine (Gibco), penicillin (50 U/ml, Gibco) and streptomycin (50 μg/ml, Gibco). A549 and A427 cell lines were obtained from American Type Culture Collection, and cells were profiled for authentication (Eurofins Genomics, Luxembourg). Cells were tested to be free of mycoplasma contaminations twice a year. For experiments, cells were harvested at ~70% confluency, and the number of viable cells was determined using a CASY counter (Roche, Basel, Switzerland).
10
Assessing Candida auris Adhesion Capacity
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C. auris isolates were grown to the logarithmic phase in YPD medium at 30°C and counted on a CASY counter (Roche). Cells were then diluted to 1 × 106 cells/mL in YPD medium, and aliquots of 100 μL of this dilution were distributed into a well of a 96-well polystyrene plate (tissue culture treated; CytoOne). The plate was then incubated in a static incubator at 37°C for 4 h, and the adhered fungal biomass was subsequently quantified using crystal violet. Briefly, YPD medium was removed by inverting the plate and tapping it on paper towels. The wells were then washed three times with phosphate-buffered saline (PBS) and incubated with 100 μL methanol (Merck) for 15 min. The plates were left to dry overnight in a chemical safety cabinet, followed by staining with 100 μL 0.1% crystal violet for 5 min and three washes with dH2O. Crystal violet was dissolved from the stained biomass by adding 100 μL of 33% acetic acid and by plate shaking for 1 min at 800 rpm. The supernatants of dissolved crystal violet were transferred into fresh wells of a 96-well plate, and the absorbance at 590 nm was recorded using a Victor Nivo plate reader (PerkinElmer).
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