For calibration, the culture depth previously calculated was used. A computer program [ 32 ] was used to generate random paths along which images were taken. For this procedure hereinafter referred to as the Neubauer methodwe used a Neubauer Improved glass counting chamber Electron Microscopy Sciences, Hatfield, PA with a grid of perpendicular lines etched in the middle region Additional file 1 : Figure S1.
To allow for comparisons between automated, field and Neubauer counting, we made the three counting efforts similar by adjusting the number of Neubauer cells to be counted. With automated and field counting, a total volume of 3. To count a similar volume with a Neubauer chamber, we calculated the number of Neubauer large squares Additional file 1 : Figure S1 to be counted by dividing 3. To fill each chamber cell, the content of each culture dish was poured into a 15 ml conical flask.
To avoid inter-counter variation, all counting was performed by the same operator. Flow cytometry allows for the fast detection and quantification of fluorescent particles when they are excited with a fluorescent light source.
Usually, cells are stained with a fluorescent dye to make them fluorescent. We used three flow cytometry methods based on the detection of 1 microspore autofluorescence, 2 propidium iodide PI -stained microspores, and 3 side scattered light SSCwhich is proportional to the overall size, granularity and internal complexity of the measured particle [ 33 ] with no need for fluorescence emission. For direct counting of unstained microspores, the contents of culture dishes were resuspended and loaded into plastic vials and directly charged into the loading port.
The suspension was loaded in the flow cytometer, which provided the number of fluorescent counts recorded, their fluorescent intensity, and the volume where the counts were recorded. For cell counting, 1 ml of microspore suspension was taken from each culture dish. The suspension was incubated with 0. The stained suspension was loaded in the flow cytometer, which provided the number of fluorescent counts recorded, their fluorescent intensity, and the volume where the counts were recorded.
The data obtained were multiplied by 2 to be comparable with those of other methods. Vials were directly charged into the loading port. To test to what extent sedimentation of microspores at the bottom of the vial, where the aspiration system cannot reach, could be reducing the number of counted microspores, we counted the cells suspended in the vial, washed the vial thrice with one additional ml of culture medium to resuspend the potentially sedimented microspores, counted the cells of each washing medium, and then calculated the density by dividing the total number of microspores counted in all rounds including washings by the initial volume where microspores were suspended excluding washings.
For all flow cytometry-based methods and cultures, three different samples were taken and processed, their individual densities calculated and then averaged. For all methods, particle densities were calculated dividing the total number of counts by the volume loaded for these counts. In mixed samples, fluorosphere and microspore densities were individually calculated using SSC counts from all the peaks obtained. After each counting round, the whole system was thoroughly cleaned to prevent wrong counts from previous samples.
Reproducibility: the similarity of data obtained with the same method from samples in different conditions. In addition, we defined concordance as the agreement of measurements obtained with different methods.
Accuracy was assessed by calculating the percentage of deviation of each individual measurement from the theoretical for microspores or real value for fluorospheres. For a visual and easy-to-understand representation of accuracy and precision results, box and whiskers graphs were developed for each method and group of samples.
In all cases, repeated measurements were summarized by their mean and standard deviation. To assess the reproducibility of each method, two different measurements were performed in the same cultures, the first at days 3 and the second at days Concordance among methods was assessed using pairwise Bland—Altman plots [ 34 ].
Statistical analysis and charts were performed with Conduit Conversion - Cellgraft - Cellgraft (CD) R software version 3. For evaluation of precision, accuracy, reproducibility and concordance between methods, a total of 17 microspore cultures were performed and measured using the different methods tested in this work.
Microspore density was estimated for each culture at days 3 and Conduit Conversion - Cellgraft - Cellgraft (CD), this culture stage is still too early to detect important morphological differences between them.
Thus, in terms of morphology, cultures at this stage are characterized by the presence of regular eggplant microspores, identical to those present in the anther, and microspores swollen as a consequence of the androgenic switch that makes them to enlarge within the exine arrows in Fig. In turn, 18 day-old cultures present enlarged microspores or microspore-derived embryos, produced as a consequence of cell divisions within the exine arrows in Fig.
These different developmental fates imply size increases only in embryogenic and pollen-like microspores, but the total number initially inoculated in culture dishes remains unchanged. In turn, suspended fluorospheres Fig.
Cultures at this stage are principally composed of regular eggplant microspores together with few slightly enlarged microspores arrows. Cultures at this stage are principally composed of regular eggplant microspores together with few enlarged microspores or microspore-derived embryos arrows.
These particles are very regular in size and shape. As a preliminary step to be confident with all the analysis performed, we checked the precision of the pipettes used in this work. Therefore, we assumed that our pipettes were well calibrated, and therefore valid for this study. We calculated the mean, standard deviation, median and 1st and 3rd quartiles of measurements performed with each method Table 1. Figure 2 a shows a graphical representation of the measurements in cultures at days 3 and Next, we performed five independent measurements of fluorosphere suspensions with each method, using the undiluted, and dilutions.
Results are shown in Table 2 and represented in Fig. All the means were below the theoretical or real density. In terms of precision, the methods based on flow cytometry showed the highest values lowest dispersion and the Neubauer method showed the highest dispersion of data in microspore measurements Fig.
Nevertheless, the Neubauer method showed a high precision in fluorosphere measurements. The precision of methods that count cells from images taken Conduit Conversion - Cellgraft - Cellgraft (CD) the dish automated and manual-counting methods presented the lowest values. Results were remarkably closer to the assumed reference value when undiluted suspensions were measured with the Neubauer method, deviating only In undiluted suspensions, some methods seemed to deviate from the expected value more than at higher dilutions, suggesting that their accuracy may depend on particle density Fig.
That was the case of image-dependent methods manual-counting and automatic counter methods. Dashed lines represent the expected microspore in a and fluorosphere densities in b. Note that values in B are expressed as neperian logarithms of mean fluorosphere densities. See text for further details.
The use of flow cytometry to measure autofluorescence of unstained microspores was the method that showed the worst performance for all parameters tested Table 1. In an attempt to find out the cause of such a discrepancy, we speculated that it could be due to sedimentation of microspores at the bottom of the vial, where the aspiration system cannot reach.
However, the results of these experiments were not different from those without washings data not shown. Therefore, we concluded that microspore autofluorescence was not sufficiently high or homogeneous to detect all the microspores passed through the flow cytometer. As a consequence, we decided to discard flow cytometry with unstained microspores for further experiments. For the analysis of the reproducibility of methods, we compared measurements in samples at two different moments of culture progression days 3 and Differences between measurements for each method are depicted by Bland—Altman plots in Fig.
As a formal measure, the coefficient of repeatability CR was obtained for each method Table 3 using data of these two different conditions. Moreover, we performed an ANOVA analysis comparing data from both days 3 and 18 where none of the methods showed significant differences Table 3.
The Neubauer method Fig. Finally, field counting Fig. Bland—Altman comparisons of reproducibility of each method by comparing 3 and 18 day-old microspore culture data. Difference values of the Y axis are expressed in thousands. In order to analyze the level of agreement between methods, we performed Bland—Altman plots from microspore density measurements Fig.
However, comparisons between Neubauer and cytometry-based methods Fig. Thus, despite their good level of agreement, flow cytometry methods seem to induce a non-negligible underestimation, at least, with respect to Neubauer method.
In general, automated counter and field counting methods showed low concordance with the rest of methods Fig. Fluorosphere measurements were not used for this comparison because they have a known, real value to compare with. Bland—Altman comparisons of agreement between methods. Using it, we obtained results similar to those of the Bland—Altman plots. In general, microspore counts showed in all cases values higher than fluorosphere counts.
Concordances between methods were minimal when high fluorosphere concentrations were used, with concordance coefficient values near to zero. The best levels of agreement were found in comparisons between flow cytometry-based methods concordance coefficient ranging from 0. The Neubauer method showed moderate concordance values when compared to flow cytometry methods from 0. The lowest level of concordance in microspore counts was found when the field counting method was compared with the Neubauer, PI and SSC.
All these results revealed a positive bias of the Neubauer method with respect to most Conduit Conversion - Cellgraft - Cellgraft (CD) the other methods Fig. Then, we checked culture densities at 3 and day old cultures using each of the four methods, as usual.
As seen in Fig. When the automated cell counter was used for the initial adjustment, all counts with the exception of the automated counter were above , being notably higher in the case of the Neubauer method An initial adjustment with the field counting method resulted in values aroundwhen counted with the Neubauer method, but 8.
When cultures were initially adjusted with the flow cytometer, counts at days 3 with all four methods revolved around , but they were clearly below at days 18, except for the Neubauer method. Nevertheless, a tendency to underestimate its own initial count was found for flow cytometry at both timepoints.
From these results, we could conclude that the automated counter, field counting and flow cytometry tended to underestimate densities, since when they were used to adjust the initial density, all other methods yielded higher counts at both timepoints. This was specially dramatic in the case of flow cytometry and field counting, which at days 3 and 18 yielded counts lower than in the initial adjustment made using the same methods.
As to the Neubauer method, it could be thought that it tends to overestimate densities, since in general, this was the method yielding highest counts. Comparison of microspore density measurements performed at days 3 a18 b with four counting methods in cultures whose initial microspore density was adjusted to The common use of fluorospheres to estimate cell densities implies their use as internal standards mixed with cell suspensions, usually for flow cytometry.
In our study, we also tried to estimate microspore density from a known quantity of fluorospheres mixed with cells. Measurements were performed with Neubauer, automated counter, field counting and SSC method.
Results of these assays Conduit Conversion - Cellgraft - Cellgraft (CD) shown in Table 5. The automated counter image detection system was unable to differentiate between fluorospheres smaller and microspores largerso we could not obtain any estimation in this case. Thus, we concluded that at least for microspores, this method, although fast and straightforward, is not accurate enough, at least when used with non flow cytometry methods. Indeed, the best results were obtained with the latter, which is reasonable since this counting strategy has been designed for flow cytometry.
For most cell culture systems, optimal cell progression depends on the optimization of the initial cell density at the onset of the culture. A paradigmatic example of this is isolated microspore culture, where the developmental switch relies on the successful optimization of many different experimental parameters that critically affect the efficiency of the process, and one of them is the density at which microspores are suspended in liquid medium.
It affects not only the efficiency of the induction of microspores towards embryogenesis, but also a successful conversion of induced microspores into viable, germinating embryos [ 3510163738 ].
Due to the importance of this initial step, not only for microspore culture but for virtually all animal and plant cell cultures, in this work we compared the use of different methods that have been used or could potentially be used to calculate particle densities using two different particles: eggplant microspores and fluorospheres. In light of our results, we can divide the methods used in three groups: flow cytometry methods, automated counter and field counting, and Neubauer chamber.
Each has both positive and negative aspects. They are summarized in Table 6 and discussed below. We evaluated three different methods based on the use of flow cytometry. The first method consisted on the analysis of unstained microspores, assuming that the natural autofluorescence of the exine coat could be sufficiently high to be detected and quantified by the system.
However, the analysis of seven 3-day old cultures was enough to realize that this method presented serious limitations. Obviously, we strongly discourage its use. Nevertheless, evaluation of accuracy using a standard with a known concentration and precision with a high number of measurements lead us to conclude that cytometry methods are not as accurate and precise as the rest.
In addition, they repeatedly showed a negative bias with respect to other methods, specially the Neubauer method. Thus, a question arises as to why the flow cytometry methods we used have such a low performance. The use of fluorescent beads is a well-known method to calculate cell densities through flow cytometry, at least for animal cell cultures [ 3940 ]. Our work is not the only one showing a consistent bias either positive or negative of the Neubauer method compared to others [ 41 ].
However, other studies have compared hemacytometers versus automated counting methods in animal cells [ 12394042434445 ], and no significant bias has been reported. After a thorough study of the different user-based technical factors that could potentially cause such a bias including bad calculations, wrong chamber dimensions, pipetting errors, uneven cell distribution, contamination, user-to-user variation, and filling problems, among otherswe found a possible cause that might explain such a bias.
Several studies comparing methods to calculate the flow rate of flow cytometer fluidics systems, have acknowledged the limitations of many flow cytometers to perform a proper estimation of the volume loaded [ 4647 ], which may preclude a right estimation of particle densities. This led us to evaluate the volume estimation accuracy of our flow cytometer and, as expected, the effective volume loaded never coincided with the volume estimated by the device data now shown.
This could surely explain the negative bias and the low accuracy and precision of the flow cytometry-based methods we used. These have the disadvantage that compared to conduit fill apps do not offer the flexibility of calculating different wire gauges and other perks, and can be slow and error-prone. You can confirm this value using the online conduit fill calculator on our home page.
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