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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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Measured versus calculated concentration of nuclei from INS-1 cells counted by all three methods. (A) All data plotted on log-log coordinates. (B) Data for concentrations up to 106 nuclei/ml plotted on linear coordinates. (C) Data for concentrations up to 2 × 105 nuclei/ml plotted on linear coordinates. Data for visual counting with a hemacytometer were fitted with a straight line through the origin and yielded a slope of 1.11 ± 0.03. To determine calculated concentration, each measured concentration was multiplied by its dilution factor to estimate the original stock solution concentration. The average of these estimates in the range of 1–5 × 105 (flow cytometry) and 2–7 × 104 nuclei/ml (aperture resistance) agreed within 2%. The combined average was taken as the actual stock nuclei concentration, from which all other values were calculated using the known dilution factors. Line of identity is dashed.
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Figure 2: Measured versus calculated concentration of nuclei from INS-1 cells counted by all three methods. (A) All data plotted on log-log coordinates. (B) Data for concentrations up to 106 nuclei/ml plotted on linear coordinates. (C) Data for concentrations up to 2 × 105 nuclei/ml plotted on linear coordinates. Data for visual counting with a hemacytometer were fitted with a straight line through the origin and yielded a slope of 1.11 ± 0.03. To determine calculated concentration, each measured concentration was multiplied by its dilution factor to estimate the original stock solution concentration. The average of these estimates in the range of 1–5 × 105 (flow cytometry) and 2–7 × 104 nuclei/ml (aperture resistance) agreed within 2%. The combined average was taken as the actual stock nuclei concentration, from which all other values were calculated using the known dilution factors. Line of identity is dashed.

Mentions: INS-1 cells were suspended and disrupted to form a stock nuclei suspension. Aliquots diluted to cover a wide concentration range were counted by each method. Measured nuclei concentration is plotted in Figure 2A as a function of the calculated concentration over four orders of magnitude and in Figures 2B and 2C on linear coordinates over different concentration ranges to illustrate where each counting method departs from linearity. Concentration measured with a hemacytometer varied linearly with calculated concentration over the entire range but was uniformly about 11% higher than the line of identity (Figure 2C). Flow cytometry and aperture electrical resistance data agreed well with the line of identity up to about 5 × 105 and 7 × 107 nuclei/ml, respectively (Figure 2B), above which the data began to deviate from linearity. These deviations arose from coincident events (two nuclei registering in the sensing zone at the same time), which increased with concentration. The Coulter Multisizer II gave erroneously high readings at less than about 104 nuclei/ml (data not shown).


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)

Measured versus calculated concentration of nuclei from INS-1 cells counted by all three methods. (A) All data plotted on log-log coordinates. (B) Data for concentrations up to 106 nuclei/ml plotted on linear coordinates. (C) Data for concentrations up to 2 × 105 nuclei/ml plotted on linear coordinates. Data for visual counting with a hemacytometer were fitted with a straight line through the origin and yielded a slope of 1.11 ± 0.03. To determine calculated concentration, each measured concentration was multiplied by its dilution factor to estimate the original stock solution concentration. The average of these estimates in the range of 1–5 × 105 (flow cytometry) and 2–7 × 104 nuclei/ml (aperture resistance) agreed within 2%. The combined average was taken as the actual stock nuclei concentration, from which all other values were calculated using the known dilution factors. Line of identity is dashed.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Measured versus calculated concentration of nuclei from INS-1 cells counted by all three methods. (A) All data plotted on log-log coordinates. (B) Data for concentrations up to 106 nuclei/ml plotted on linear coordinates. (C) Data for concentrations up to 2 × 105 nuclei/ml plotted on linear coordinates. Data for visual counting with a hemacytometer were fitted with a straight line through the origin and yielded a slope of 1.11 ± 0.03. To determine calculated concentration, each measured concentration was multiplied by its dilution factor to estimate the original stock solution concentration. The average of these estimates in the range of 1–5 × 105 (flow cytometry) and 2–7 × 104 nuclei/ml (aperture resistance) agreed within 2%. The combined average was taken as the actual stock nuclei concentration, from which all other values were calculated using the known dilution factors. Line of identity is dashed.
Mentions: INS-1 cells were suspended and disrupted to form a stock nuclei suspension. Aliquots diluted to cover a wide concentration range were counted by each method. Measured nuclei concentration is plotted in Figure 2A as a function of the calculated concentration over four orders of magnitude and in Figures 2B and 2C on linear coordinates over different concentration ranges to illustrate where each counting method departs from linearity. Concentration measured with a hemacytometer varied linearly with calculated concentration over the entire range but was uniformly about 11% higher than the line of identity (Figure 2C). Flow cytometry and aperture electrical resistance data agreed well with the line of identity up to about 5 × 105 and 7 × 107 nuclei/ml, respectively (Figure 2B), above which the data began to deviate from linearity. These deviations arose from coincident events (two nuclei registering in the sensing zone at the same time), which increased with concentration. The Coulter Multisizer II gave erroneously high readings at less than about 104 nuclei/ml (data not shown).

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