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Horseradish peroxidase as a label of injured cells
Abstract:
The present study is concerned with artifacts likely to occur in a horseradish peroxidase exclusion test. Incubation of murine peritoneal macrophages and lymphocytes with the peroxidase showed a close relationship between the number of living cells and the percentage of cells excluding the tracer. The penetration of the cytoplasm by horseradish peroxidase is attributed to an increase in the permeability of the cell membrane during the incubation (ranging from 10 to 120 min). It was not increased by the presence of tracer throughout the incubation period. However, concomitant fixation of the cell in the presence of horseradish peroxidase caused an increase in the influx of the tracer. The horseradish peroxidase exclusing test applied to the guinea-pig organ of Corti has proved to be valid provided that: (a) mechanical lesions prior to the tracer incubation are avoided; (b) incubation is terminated by removal of the extracellular tracer; (c) fixation is carried out as soon as possible; (d) a low concentration of horseradish peroxidase is used; and (e) specimens are incubated in diaminobenzidine-H2O2 medium for the shortest possible period. Although fixation-induced cytoplasmic infiltration by horseradish peroxidase was not detected in cochlear specimens, the findings call attention to possible sources of error and define the level of significance of the test. Horseradish peroxidase does not appear to be a cytotoxic agent under the conditions used.
Insights
The horseradish peroxidase exclusion test is valid for cell viability assessment, but artifacts can occur. Proper procedures, including avoiding mechanical damage and prompt fixation, are crucial for accurate results.
Area of Science:
- Cell Biology
- Histology
- Biochemistry
Background:
- The horseradish peroxidase (HRP) exclusion test is commonly used to assess cell membrane integrity and viability.
- Potential artifacts can compromise the accuracy of HRP exclusion tests, particularly in sensitive tissues.
Purpose of the Study:
- To investigate potential artifacts in horseradish peroxidase exclusion tests.
- To determine the validity and optimal conditions for using HRP exclusion tests in murine macrophages, lymphocytes, and guinea-pig cochlear specimens.
Main Methods:
- Incubation of murine peritoneal macrophages and lymphocytes with HRP.
- Application of HRP exclusion test to guinea-pig organ of Corti.
- Evaluation of factors influencing HRP penetration, including incubation time, fixation, and tracer concentration.
Main Results:
- A strong correlation was observed between the number of viable cells and the percentage of cells excluding HRP.
- Increased cell membrane permeability during incubation (10-120 min) contributed to HRP cytoplasmic penetration.
- Concomitant fixation with HRP increased tracer influx, while prolonged tracer presence did not.
- The HRP exclusion test was deemed valid for the organ of Corti under specific conditions, including avoiding mechanical lesions, prompt fixation, and using low HRP concentrations.
Conclusions:
- The horseradish peroxidase exclusion test can be a valid measure of cell viability when specific procedural guidelines are followed.
- Careful attention to fixation timing, mechanical handling, and HRP concentration is essential to prevent artifacts.
- Horseradish peroxidase was not found to be cytotoxic under the tested conditions.