Comparison of six different methods to calculate cell densities

Camacho-Fernández, Carolina; Hervás, David Sánchez; Rivas-Sendra, Alba; Marín, Ma Pilar; Seguí-Simarro, José M.

doi:10.1186/s13007-018-0297-4

Cited by 50 publications

(28 citation statements)

References 44 publications

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“…The viability of separated leukocytes (>95%) was evaluated by dye exclusion (propidium iodide; 2 μg/mL, Calbiochem, Germany). For absolute counting of total leukocytes, blood was diluted 1:2 in PBS and was then mixed with Türk Solution (Sigma-Aldrich, USA), and the mixture was poured onto the Neubauer counting chamber [ 26 ]. Cells were counted in four large squares under a microscope, and total leukocyte count was calculated (cell/μL blood).…”

Section: Methodsmentioning

confidence: 99%

Leukocyte populations and their cell adhesion molecules expression in newborn dromedary camel calves

2020

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Background and Aim: Different properties of the newborn immune system have been characterized in many species. For the newborn camel calf, however, the phenotype and composition of blood leukocytes have so far not been evaluated. The current study aimed to analyze the distribution of leukocyte subpopulations and their expression pattern of cell adhesion molecules in newborn and adult dromedary camels. Materials and Methods: Blood samples were collected from 17 newborn camel calves and 32 adult camels. For each sample, total leukocytes were separated and analyzed for their composition and cell adhesion molecules expression by flow cytometry. Results: In comparison to adult camels, newborn camel calves had higher leukocyte numbers and higher numbers of neutrophils, monocytes, and lymphocytes but lower numbers of eosinophils in their blood. Among the lymphocyte populations in calves, the fractions of B cells and γδ T cells were elevated when compared to adults, whereas CD4-positive T cells were reduced. The comparison between camel calves and adult camels revealed significantly lower expression of the cell adhesion molecules CD11a, CD11b, and CD18 on granulocytes, monocytes, and lymphocytes in calves. Conclusion: Newborn camel calves show a distinct composition and phenotype pattern of blood leukocytes when compared to adult camels. The observed rise in many leukocyte populations in calf blood may be due to reduced migratory activity in calf leukocyte populations.

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Section: Methodsmentioning

confidence: 99%

Leukocyte populations and their cell adhesion molecules expression in newborn dromedary camel calves

2020

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“…Microscopic counting of leukocytes: Whole blood was diluted 1: 4 in PBS and was then mixed with Türk's solution (final dilution 1:20; Merck Millipore) and 10 µl of the mixture was poured onto the hemocytometer (Neubauer cell counter) for counting under the microscope. Leukocytes (in blue color were counted in four big squares of the cell counter and total leukocyte count was calculated (Camacho-Fernandez et al, 2018).…”

Section: Research Article Materials and Methodsmentioning

confidence: 99%

Leukocytes Immunophenotype and Phagocytosis Activity in Pregnant and Nonpregnant Dromedary She Camels

Hussen¹

2020

PVJ

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Pregnancy is associated with different local and systemic changes in immune cell phenotype and function. Here, we used flow cytometry and membrane immunofluorescence for the comparison between pregnant (n=18) and non-pregnant she camels (n=21) in terms of immunophenotype of blood leukocytes and phagocytosis activity of neutrophils. Camel blood leukocytes were labeled with monoclonal antibodies to camel antigens and analyzed by flow cytometry. Pregnant she camels displayed higher leukocyte counts than non-pregnant animals. Differential analysis of leukocyte populations revealed higher proportions of monocytes, lymphocytes and CD4-positive T cells but lower proportion of neutrophils in pregnant she camels in comparison to non-pregnant animals. Pregnancy was also associated with enhanced expression of the cell adhesion molecule CD11a on neutrophils but reduced CD11a expression on lymphocytes in pregnant animals. After incubation with FITC-labeled bacteria, flow cytometric exvivo analysis of bacterial phagocytosis by neutrophils showed higher phagocytosis capacity in neutrophils from pregnant than from non-pregnant animals. Together these results indicate significant modulating effects of pregnancy on the immune system of she camels.

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“…To evaluate the performance of the automatic methods and to compare them with the standard manual method, the concepts of accuracy, precision, reproducibility, concordance and bias were evaluated [ 18 , 61 ]. Data from the optimization of lighting conditions were analysed by a Generalized Linear Model (GLM).…”

Section: Methodsmentioning

confidence: 99%

Saving time maintaining reliability: a new method for quantification of Tetranychus urticae damage in Arabidopsis whole rosettes

et al. 2020

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Background The model species Tetranychus urticae produces important plant injury and economic losses in the field. The current accepted method for the quantification of the spider mite damage in Arabidopsis whole rosettes is time consuming and entails a bottleneck for large-scale studies such as mutant screening or quantitative genetic analyses. Here, we describe an improved version of the existing method by designing an automatic protocol. The accuracy, precision, reproducibility and concordance of the new enhanced approach are validated in two Arabidopsis accessions with opposite damage phenotypes. Results are compared to the currently available manual method. Results Image acquisition experiments revealed that the automatic settings plus 10 values of brightness and the black background are the optimal conditions for a specific recognition of spider mite damage by software programs. Among the different tested methods, the Ilastik-Fiji tandem based on machine learning was the best procedure able to quantify the damage maintaining the differential range of damage between accessions. In addition, the Ilastik-Fiji tandem method showed the lowest variability within a set of conditions and the highest stability under different lighting or background surroundings. Bland-Altman concordance results pointed out a negative value for Ilastik-Fiji, which implies a minor estimation of the damage when compared to the manual standard method. Conclusions The novel approach using Ilastik and Fiji programs entails a great improvement for the quantification of the specific spider mite damage in Arabidopsis whole rosettes. The automation of the proposed method based on interactive machine learning eliminates the subjectivity and inter-rater-variability of the previous manual protocol. Besides, this method offers a robust tool for time saving and to avoid the damage overestimation observed with other methods.

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Comparison of six different methods to calculate cell densities

Cited by 50 publications

References 44 publications

Leukocyte populations and their cell adhesion molecules expression in newborn dromedary camel calves

Leukocyte populations and their cell adhesion molecules expression in newborn dromedary camel calves

Leukocytes Immunophenotype and Phagocytosis Activity in Pregnant and Nonpregnant Dromedary She Camels

Saving time maintaining reliability: a new method for quantification of Tetranychus urticae damage in Arabidopsis whole rosettes

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