Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death
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Bibliographic record
Abstract
Studies of cellular apoptosis have been significantly impacted since the introduction of flow cytometry-based methods. Propidium iodide (PI) is widely used in conjunction with Annexin V to determine if cells are viable, apoptotic, or necrotic through differences in plasma membrane integrity and permeability. The Annexin V/PI protocol is a commonly used approach for studying apoptotic cells. PI is used more often than other nuclear stains because it is economical, stable and a good indicator of cell viability, based on its capacity to exclude dye in living cells. The ability of PI to enter a cell is dependent upon the permeability of the membrane; PI does not stain live or early apoptotic cells due to the presence of an intact plasma membrane. In late apoptotic and necrotic cells, the integrity of the plasma and nuclear membranes decreases, allowing PI to pass through the membranes, intercalate into nucleic acids, and display red fluorescence. Unfortunately, we find that conventional Annexin V/ PI protocols lead to a significant number of false positive events (up to 40%), which are associated with PI staining of RNA within the cytoplasmic compartment. Primary cells and cell lines in a broad range of animal models are affected, with large cells (nuclear: cytoplasmic ratios <0.5) showing the highest occurrence. Herein, we demonstrate a modified Annexin V/ PI method that provides a significant improvement for assessment of cell death compared to conventional methods. This protocol takes advantage of changes in cellular permeability during cell fixing to promote entry of RNase A into cells following staining. Both the timing and concentration of RNase A have been optimized for removal of cytoplasmic RNA. The result is a significant improvement over conventional Annexin V/ PI protocols (< 5% events with cytoplasmic PI staining).
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Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from it