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Continuous and long-term volume measurements with a commercial Coulter counter.

ABSTRACT: We demonstrate a method to enhance the time resolution of a commercial Coulter counter and enable continuous and long-term cell size measurements for growth rate analyses essential to understanding basic cellular processes, such as cell size regulation and cell cycle progression. Our simple modifications to a commercial Coulter counter create controllable cell culture conditions within the sample compartment and combine temperature control with necessary adaptations to achieve measurement stability over several hours. We also wrote custom software, detailed here, to analyze instrument data files collected by either this continuous method or standard, periodic sampling. We use the continuous method to measure the growth rate of yeast in G1 during a prolonged arrest and, in different samples, the dependency of growth rate on cell size and cell cycle position in arrested and proliferating cells. We also quantify with high time resolution the response of mouse lymphoblast cell culture to drug treatment. This method provides a technique for continuous measurement of cell size that is applicable to a large variety of cell types and greatly expands the set of analysis tools available for the Coulter counter.

SUBMITTER: Bryan AK

PROVIDER: S-EPMC3260162 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Continuous and long-term volume measurements with a commercial Coulter counter.

Bryan Andrea K AK Engler Alex A Gulati Amneet A Manalis Scott R SR

PloS one 20120117 1

We demonstrate a method to enhance the time resolution of a commercial Coulter counter and enable continuous and long-term cell size measurements for growth rate analyses essential to understanding basic cellular processes, such as cell size regulation and cell cycle progression. Our simple modifications to a commercial Coulter counter create controllable cell culture conditions within the sample compartment and combine temperature control with necessary adaptations to achieve measurement stabil ...[more]

PMID: 22272256

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Project description:BackgroundThe malarial parasite Plasmodium falciparum is an auxotroph for purines, which are required for nucleic acid synthesis during the intra-erythrocytic developmental cycle (IDC) of the parasite. The capabilities of the parasite and extent to which it can use compensatory mechanisms to adapt to purine deprivation were studied by examining changes in its metabolism under sub-optimal concentrations of hypoxanthine, the primary precursor utilized by the parasite for purine-based nucleic acid synthesis.MethodsThe concentration of hypoxanthine that caused a moderate growth defect over the course of one IDC was determined. At this concentration of hypoxanthine (0.5 μM), transcriptomic and metabolomic data were collected during one IDC at multiple time points. These data were integrated with a metabolic network model of the parasite embedded in a red blood cell (RBC) to interpret the metabolic adaptation of P. falciparum to hypoxanthine deprivation.ResultsAt a hypoxanthine concentration of 0.5 μM, vacuole-like structures in the cytosol of many P. falciparum parasites were observed after the 24-h midpoint of the IDC. Parasites grown under these conditions experienced a slowdown in the progression of the IDC. After 72 h of deprivation, the parasite growth could not be recovered despite supplementation with 90 µM hypoxanthine. Simulations of P. falciparum metabolism suggested that alterations in ubiquinone, isoprenoid, shikimate, and mitochondrial metabolism occurred before the appearance of these vacuole-like structures. Alterations were found in metabolic reactions associated with fatty acid synthesis, the pentose phosphate pathway, methionine metabolism, and coenzyme A synthesis in the latter half of the IDC. Furthermore, gene set enrichment analysis revealed that P. falciparum activated genes associated with rosette formation, Maurer's cleft and protein export under two different nutrient-deprivation conditions (hypoxanthine and isoleucine).ConclusionsThe metabolic network analysis presented here suggests that P. falciparum invokes specific purine-recycling pathways to compensate for hypoxanthine deprivation and maintains a hypoxanthine pool for purine-based nucleic acid synthesis. However, this compensatory mechanism is not sufficient to maintain long-term viability of the parasite. Although P. falciparum can complete a full IDC in low hypoxanthine conditions, subsequent cycles are disrupted.

Project description:To assess the long-term prognostic value of an integral of longitudinal measurements of plasma neurofilament light chain levels (NfLlong) over 12 and 24 months vs single neurofilament light chain (NfL) measurements in patients with relapsing-remitting MS (RRMS) and its additional value when combined with clinical and MRI measures. This analysis included continuously fingolimod-treated patients with RRMS from the 24-month FTY720 Research Evaluating Effects of Daily Oral therapy in Multiple Sclerosis (FREEDOMS)/12-month Trial Assessing Injectable Interferon vs FTY720 Oral in Relapsing-Remitting Multiple Sclerosis (TRANSFORMS) phase 3 trials and their long-term extension, LONGTERMS. Patients were classified into high (≥30 pg/mL, n = 110) and low (<30 pg/mL, n = 164) NfL categories based on the baseline (BL) NfL value or the geometric mean NfLlong calculated over 12 and 24 months to predict disability-related outcomes and brain volume loss (BVL). The additional prognostic value of NfL was quantified using the area under the receiver operating characteristic (ROC) curve. A single high (vs low) NfL measure at BL was prognostic of a higher risk of reaching Expanded Disability Status Scale (EDSS) score ≥4 earlier (hazard ratio [HR] = 2.19; 95% CI = 1.21-3.97) and higher BVL over 120 months (difference: -1.12%; 95% CI = -2.07 to -0.17). When NfLlong was measured over 24 months, high NfL was associated with a higher risk of reaching EDSS score ≥4 (HR = 7.91; 95% CI = 2.99-20.92), accelerated 6-month confirmed disability worsening (HR = 3.14; 95% CI = 1.38-7.11), and 20% worsening in the Timed 25-Foot Walk Test (HR = 3.05; 95% CI = 1.38-6.70). Area under the ROC curve was consistently highest in models combining NfL with clinical and MRI measures. NfLlong had a higher prognostic value than single NfL assessments on long-term outcomes in RRMS. Combining it with clinical and MRI measures increased sensitivity and specificity to predict long-term disease outcomes. This study provides Class I evidence that NfLlong was more strongly associated with long-term outcomes than single NfL assessments in patients with RRMS.

Dataset Information

Continuous and long-term volume measurements with a commercial Coulter counter.

Publications

Continuous and long-term volume measurements with a commercial Coulter counter.

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