EFFECT OF MULTISTAGE ISOLATION OF NEUTROPHILS ON THEIR NUMBER AND VIABILITY
Despite the numerous methods available for isolating neutrophils from peripheral blood, the problem of obtaining a sufficient quantity of viable cells with high purity for quantitatively determining neutrophil cytokines and their mRNA expression remains actual. Recommended multi-step purification me...
| Main Authors: | , , |
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| Format: | Article |
| Language: | Russian |
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St. Petersburg branch of the Russian Association of Allergologists and Clinical Immunologists
2019-08-01
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| Series: | Медицинская иммунология |
| Subjects: | |
| Online Access: | https://www.mimmun.ru/mimmun/article/view/2948 |
| Summary: | Despite the numerous methods available for isolating neutrophils from peripheral blood, the problem of obtaining a sufficient quantity of viable cells with high purity for quantitatively determining neutrophil cytokines and their mRNA expression remains actual. Recommended multi-step purification methods significantly prolong the isolation process, potentially leading to cell activation or apoptosis and resulting in significant cell loss. The most critical stage, in terms of duration and the number of additional manipulations with cells, is the preliminary purification of neutrophils. Addressing this current challenge, our study aimed to compare various methods of preliminary neutrophil isolation to select the most optimal one for obtaining a sufficient number of viable peripheral neutrophils.We studied the effects of three different protocols for the preliminary isolation of cell suspensions (centrifugation of whole blood on a single density gradient followed by sedimentation of red blood cells with dextran; centrifugation of whole blood on a double density gradient; rapid isolation of leukocytes using a reagent that promotes red blood cell aggregation) on the number and viability of neutrophils, purified at the final stage using negative immunomagnetic selection. Our study established that the method used for preliminary neutrophil isolation significantly affects both the number and viability of the cells. The highest number of viable neutrophils was obtained using the traditional method of blood centrifugation on a density gradient, followed by sedimentation of red blood cells with dextran. However, the three different methods of preliminary neutrophil isolation studied did not show statistically significant differences in the quantitative yield of viable cells after immunomagnetic isolation. This suggests that researchers can choose any of these methods based on their capabilities and preferences. In summary, our findings confirm previous research indicating that the multistep process of neutrophil isolation allows for obtaining a cell suspension of high purity (>99.1%), which can be utilized in future studies of their cytokine-secreting activity. However, such multi-stage isolation significantly reduces the number of neutrophils, which can be critical for initially small blood volumes. |
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| ISSN: | 1563-0625 2313-741X |