An electronic particle counter/size analyser (Multisizer™ 3 Coulter Counter, Beckman Coulter) was used to evaluate the hemocyte concentration in hemolymph. The size frequency distribution and hemocyte concentration (number of cells per milliliter) were determined. Before sample running, 0.5 mL of hemolymph was added into 9.5 mL of Isoton® II solution to crank out the mixture, and every time 1000 µL of the mixed solution were counted.
Multisizer 3 coulter counter
Manufactured by Beckman Coulter
Sourced in United States, France
The Multisizer 3 Coulter Counter is a particle size analyzer that utilizes the Coulter principle to measure the size and count of particles suspended in an electrolyte solution. It provides accurate and reliable measurements of particle size distribution across a wide range of particle sizes.
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Nucleocounter nc 250, by ChemoMetec (2 mentions)
Facscalibur flow cytometer, by BD (2 mentions)
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Mycoalert mycoplasma detection kit, by Lonza (1 mentions)
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102 protocols using multisizer 3 coulter counter
1
Hemocyte Concentration Analysis via Coulter Counter
2
Assessing HUVEC Growth in SMC Secretome
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HUVECs were seeded in complete growth medium at a 1 × 105 cells/well in 6-well plates for 24 hr. The medium was changed to M199 supplemented with 0.5% FBS (control)-based conditioned medium from the Ad-XBP1s infected SMCs and incubated for 24 hr. Upon harvest, the cells were trypsinized to single cell suspension and subjected to cell counting using a multisizer 3 coulter counter (Beckman Coulter), according to the manufacturer’s instructions.
3
Transduction of Primary Human T Cells
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Primary human CD4+ and CD8+ T cells, purchased from the Human Immunology Core at University of Pennsylvania, were isolated from healthy volunteer donors following leukapheresis by negative selection. All specimens were collected under a protocol approved by a University Institutional Review Board, and written informed consent was obtained from each donor. T cells were cultured in R10 medium and stimulated with anti-CD3 and anti-CD28 monoclonal antibodies (mAb)-coated beads (Invitrogen). Eighteen to 24 hours after activation, human T cells were transduced using a spinoculation procedure. Briefly, 0.5 × 106 T cells were infected with a multiplicity of infection (MOI) of 2 and 5 of concentrated C4-27z and MOv19-27z vector, respectively. Mixtures of cells and vectors were centrifuged at room temperature for 90min (2,500 rpm) in a table-top centrifuge (Sorvall ST 40). Human recombinant interleukin-2 (IL-2; Novartis) was added every 2–3 days to a 100 IU/mL final concentration and a cell density of 0.5 × 106 to 1 × 106 cells/mL was maintained. Once engineered T-cell cultures appeared to rest down, as determined by both decreased growth kinetics and cell-sizing determined using the Multisizer 3 Coulter Counter (Beckman Coulter), the T cells were used for functional analysis.
4
Tumorsphere Enumeration from Single Cells
5
Microbubble Formulation and Characterization
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Microbubbles were prepared from perfluorobutane gas and stabilized with a monolayer of distearoyl phosphatidylcholine and PEG stearate. 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC; Avanti Polar Lipids, Alabama, USA) and polyoxyethylene stearate (PEG40; Sigma, St. Louis, MO, USA) were dissolved in glycerol (10 mg/mL) and sonicated (Decon FS200, Decon Ultrasonics Ltd., Sussex, UK) at 40 kHz in an atmosphere of perfluorobutane (F2 Chemicals Ltd., Lancashire, UK) and vials were shaken in a Vialmix at 4500 rpm (Bristol-Myers Squibb Medical Imaging, Massachusetts, USA). As the gas was dispersed in the aqueous phase, microbubbles were formed, which were stabilized with a self-assembled lipid/surfactant monolayer. Freshly made bubbles were then washed twice to remove excessive DSPC and PEG40 and stored refrigerated in sealed vials in perfluorobutane atmosphere. A Multisizer 3 Coulter Counter (Beckman Coulter Inc., Miami, FL, USA) was used to measure the particle size distribution as well as the number of particles. The average bubble concentration was 1.58 · 109 ± 0.36 · 109 and the particle range was between 1 and 10 μm. Microbubbles were diluted to a concentration of 200 · 106 with degassed NaCl.
6
Terrigenous Silt Grain Size Analysis
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Samples from ODP Site 983 were prepared for SS analysis following established protocols24 38 . Briefly, 2–4 g of bulk fine fraction (<63 μm) was treated with acetic acid and sodium carbonate to remove carbonate and biogenic silica, respectively. The residual silicate fraction was treated with Calgon and ultrasonicted for 4 min before analysis on a Beckman Coulter Multisizer 3 coulter counter. At least two replicate measurements of the arithmetic mean calculated from the differential volume of grains within the 10–63 μm terrigenous silt fraction are reported for each sample depth. The average s.d. between replicate measurements for all samples is ±0.23 μm.
7
Cell Proliferation and Viability Assay
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About 10,000 cells transfected with the indicated siRNAs were plated on a 6‐well plate (day 0). Starting from day 3 after plating, cells were counted every day using Beckman Multisizer 3 Coulter Counter. Cells were split to maintain a confluence < 70%.
For the experiment shown in FigEV4D , 2,000 cells transfected with the indicated siRNAs were seeded in triplicate in a 96‐well cell culture plate (Corning #3596) and four regions per well were imaged every 6 h over a period of 96 h using Incucyte® SX5 Live‐Cell Analysis System (Sartorius). The surface area occupied by the cells was calculated by Incucyte analysis software and expressed as per cent (%) cell confluency.
To measure cell viability (FigEV4E ), 10,000 cells/well transfected with the indicated siRNAs were seeded in triplicate in 48‐well plates and grown for four days. Cell viability was determined using the 1% crystal violet staining containing 20% methanol (Sigma #1092180500).
For the experiment shown in Fig
To measure cell viability (Fig
8
Amino Acid Depletion Effects on Cell Proliferation
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All cell lines were plated in six-well plates in DMEM with 10% FBS at a concentration of 20,000 cells per well with the exception of MDA-MB-468 cells, which were plated at a concentration of 40,000 cells per well. The number of cells seeded for each cell line allowed for exponential growth over the course of the assay. The following day, one six-well plate of each cell line was counted to determine the initial number of cells at the time of treatment. Cells were washed three times with PBS, and 4 ml of treatment medium was added. Treatment medium was made with 10% dialysed FBS. Medium lacking specific amino acids was made from DMEM without pyruvate or amino acids supplemented with an amino acid mix containing DMEM concentrations of amino acids without arginine, leucine or serine. Arginine, leucine or serine was added back to the medium as needed. After 4 days of treatment, final cell counts were measured using a Multisizer 3 Coulter Counter (Beckman Coulter). The following formula was used to calculate proliferation rate:
Doublings per day = [log2(final day 4 cell count /initial day 0 cell count)]/4 days
Cell size measurements were taken using a Multisizer 3 Coulter Counter at the same time as cell counts, after 4 days of treatment.
Doublings per day = [log2(final day 4 cell count /initial day 0 cell count)]/4 days
Cell size measurements were taken using a Multisizer 3 Coulter Counter at the same time as cell counts, after 4 days of treatment.
9
Measuring Cellular Protein Content
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Protein concentration was calculated by dividing total protein content by cell number and cell volume for each sample. A BCA assay was used to measure total protein content, as compared with a standard curve. At the same time, cell number and volume were measured in a parallel sample using a Multisizer 3 Coulter Counter (Beckman Coulter).
10
Cell Counting and Sizing Methods
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Cells were counted with a NucleoCounter® NC-250™ (ChemoMetec A/S, Allerod, Denmark) according to manufacturer’s instructions and cell diameters were determined with a Multisizer 3 COULTER COUNTER® (Beckman Coulter, Brea, CA, USA).
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