Fast, stable method for harvesting leukocytes for measuring intracellular magnesium

Area of Science:

• Biochemistry
• Hematology
• Analytical Chemistry

Background:

• Accurate measurement of intracellular magnesium in leukocytes is crucial for understanding cellular function and various disease states.
• Existing methods for leukocyte isolation and magnesium measurement can be complex and time-consuming.

Purpose of the Study:

• To develop and validate a novel, efficient method for isolating human blood leukocytes.
• To establish a reliable protocol for quantifying intracellular magnesium concentration within these isolated leukocytes.
• To determine baseline leukocyte magnesium levels in a healthy donor population.

Main Methods:

• A new isolation protocol involving three sequential centrifugations (1300 x g, 5 min, 22°C) and two washes, without Ficoll.
• Intracellular magnesium quantification using atomic absorption spectrophotometry.
• Protein measurement via the classic Lowry method to express magnesium concentration (μmol/g protein).

Main Results:

• The method isolates 8-28% of leukocytes with high viability (86-93%) and minimal erythrocyte contamination (0-0.025%).
• Optimal isolation conditions include centrifugal forces >1000 x g, 5-15 min centrifugation, and 2-3 washes.
• Leukocyte magnesium levels are stable at 22°C for short periods but degrade by 1% per hour.
• Mean leukocyte magnesium concentration in 98 donors was 26.4 (SD 3.9) μmol/g, with no correlation to neutrophil percentage.

Conclusions:

• The described method offers a robust and reproducible approach for leukocyte isolation and intracellular magnesium measurement.
• This technique provides a valuable tool for research in cellular metabolism and hematological disorders.
• Established baseline values offer a reference point for future clinical and research applications.