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Enumeration of islets by nuclei counting and light microscopic analysis.

Pisania A, Papas KK, Powers DE, Rappel MJ, Omer A, Bonner-Weir S, Weir GC, Colton CK - Lab. Invest. (2010)

Bottom Line: With pure rat islet preparations, precision improved with increasing counts, and samples with about ≥160 islets provided a coefficient of variation of about 6%.Total number of IE by the standard method of dithizone staining/manual counting was overestimated by about 90% compared with LM/nuclei counting for 12 freshly isolated human islet research preparations.Nuclei counting combined with islet volume fraction measurements from LM is a novel method for achieving accurate islet enumeration.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139-4307, USA.

ABSTRACT
Islet enumeration in impure preparations by conventional dithizone staining and visual counting is inaccurate and operator dependent. We examined nuclei counting for measuring the total number of cells in islet preparations, and we combined it with morphological analysis by light microscopy (LM) for estimating the volume fraction of islets in impure preparations. Cells and islets were disrupted with lysis solution and shear, and accuracy of counting successively diluted nuclei suspensions was verified with (1) visual counting in a hemocytometer after staining with crystal violet, and automatic counting by (2) aperture electrical resistance measurement and (3) flow cytometer measurement after staining with 7-aminoactinomycin-D. DNA content averaged 6.5 and 6.9 pg of DNA per cell for rat and human islets, respectively, in agreement with literature estimates. With pure rat islet preparations, precision improved with increasing counts, and samples with about ≥160 islets provided a coefficient of variation of about 6%. Aliquots of human islet preparations were processed for LM analysis by stereological point counting. Total nuclei counts and islet volume fraction from LM analysis were combined to obtain the number of islet equivalents (IEs). Total number of IE by the standard method of dithizone staining/manual counting was overestimated by about 90% compared with LM/nuclei counting for 12 freshly isolated human islet research preparations. Nuclei counting combined with islet volume fraction measurements from LM is a novel method for achieving accurate islet enumeration.

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Nuclei concentration versus cell concentration for INS-1 and βTC3 cells, both measured by visual counting with a hemacytometer. Different symbols represent different batches of cells. The dashed line is the line of identity. The fitted line through the origin has a slope of 1.07 ± 0.01. Suspensions of cells were prepared to provide a wide range of concentrations. Six aliquots were taken from each suspension for counting with the hemacytometer, three for nuclei and three for cell concentration.
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Figure 3: Nuclei concentration versus cell concentration for INS-1 and βTC3 cells, both measured by visual counting with a hemacytometer. Different symbols represent different batches of cells. The dashed line is the line of identity. The fitted line through the origin has a slope of 1.07 ± 0.01. Suspensions of cells were prepared to provide a wide range of concentrations. Six aliquots were taken from each suspension for counting with the hemacytometer, three for nuclei and three for cell concentration.

Mentions: The higher concentration measured with the hemacytometer was explored further using data from three different operators. Data analogous to that in Figure 2 from multiple batches of cells (n = 4) and rat islets (n = 3) yielded a slope of 1.16 ± 0.02, and a single measurement with human islets yielded a ratio of 1.17 ± 0.04 for nuclei counted by hemacytometer and flow cytometer. Lastly, nuclei and the cell suspension from which they were prepared were counted with a hemacytometer (Figure 3). The data for nuclei concentration versus cell concentration fit a straight line with a slope of 1.07 ± 0.02. All together, nuclei concentration measured by visual counting with a hemocytometer was high by about 7 to 17% (average of all data 12 ± 5%), presumably because of the presence of large cellular fragments, particles, and debris that appeared as nuclei by visual counting but were not registered as nuclei by the two other counting methods.


Enumeration of islets by nuclei counting and light microscopic analysis.

Pisania A, Papas KK, Powers DE, Rappel MJ, Omer A, Bonner-Weir S, Weir GC, Colton CK - Lab. Invest. (2010)

Nuclei concentration versus cell concentration for INS-1 and βTC3 cells, both measured by visual counting with a hemacytometer. Different symbols represent different batches of cells. The dashed line is the line of identity. The fitted line through the origin has a slope of 1.07 ± 0.01. Suspensions of cells were prepared to provide a wide range of concentrations. Six aliquots were taken from each suspension for counting with the hemacytometer, three for nuclei and three for cell concentration.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2966546&req=5

Figure 3: Nuclei concentration versus cell concentration for INS-1 and βTC3 cells, both measured by visual counting with a hemacytometer. Different symbols represent different batches of cells. The dashed line is the line of identity. The fitted line through the origin has a slope of 1.07 ± 0.01. Suspensions of cells were prepared to provide a wide range of concentrations. Six aliquots were taken from each suspension for counting with the hemacytometer, three for nuclei and three for cell concentration.
Mentions: The higher concentration measured with the hemacytometer was explored further using data from three different operators. Data analogous to that in Figure 2 from multiple batches of cells (n = 4) and rat islets (n = 3) yielded a slope of 1.16 ± 0.02, and a single measurement with human islets yielded a ratio of 1.17 ± 0.04 for nuclei counted by hemacytometer and flow cytometer. Lastly, nuclei and the cell suspension from which they were prepared were counted with a hemacytometer (Figure 3). The data for nuclei concentration versus cell concentration fit a straight line with a slope of 1.07 ± 0.02. All together, nuclei concentration measured by visual counting with a hemocytometer was high by about 7 to 17% (average of all data 12 ± 5%), presumably because of the presence of large cellular fragments, particles, and debris that appeared as nuclei by visual counting but were not registered as nuclei by the two other counting methods.

Bottom Line: With pure rat islet preparations, precision improved with increasing counts, and samples with about ≥160 islets provided a coefficient of variation of about 6%.Total number of IE by the standard method of dithizone staining/manual counting was overestimated by about 90% compared with LM/nuclei counting for 12 freshly isolated human islet research preparations.Nuclei counting combined with islet volume fraction measurements from LM is a novel method for achieving accurate islet enumeration.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139-4307, USA.

ABSTRACT
Islet enumeration in impure preparations by conventional dithizone staining and visual counting is inaccurate and operator dependent. We examined nuclei counting for measuring the total number of cells in islet preparations, and we combined it with morphological analysis by light microscopy (LM) for estimating the volume fraction of islets in impure preparations. Cells and islets were disrupted with lysis solution and shear, and accuracy of counting successively diluted nuclei suspensions was verified with (1) visual counting in a hemocytometer after staining with crystal violet, and automatic counting by (2) aperture electrical resistance measurement and (3) flow cytometer measurement after staining with 7-aminoactinomycin-D. DNA content averaged 6.5 and 6.9 pg of DNA per cell for rat and human islets, respectively, in agreement with literature estimates. With pure rat islet preparations, precision improved with increasing counts, and samples with about ≥160 islets provided a coefficient of variation of about 6%. Aliquots of human islet preparations were processed for LM analysis by stereological point counting. Total nuclei counts and islet volume fraction from LM analysis were combined to obtain the number of islet equivalents (IEs). Total number of IE by the standard method of dithizone staining/manual counting was overestimated by about 90% compared with LM/nuclei counting for 12 freshly isolated human islet research preparations. Nuclei counting combined with islet volume fraction measurements from LM is a novel method for achieving accurate islet enumeration.

Show MeSH
Related in: MedlinePlus