Quantitative fluorescence cytometric analysis of Bcl-2 levels in tumor cells exhibiting a wide range of inherent Bcl-2 protein expression: Correlation with Western blot analysis
Bibliographic record
Abstract
BACKGROUND: A protocol to measure a wide range of Bcl-2 protein expression using quantitative fluorescence cytometry (QFCM) in different cell types was developed for use with flow cytometry. Bcl-2 measurements obtained by flow cytometry were correlated with Western blot Bcl-2 measurements to confirm specificity of the Bcl-2-FITC staining. This protocol was applied to measure absolute levels of Bcl-2 protein in different tumor cell lines including Bcl-2-transfected breast carcinoma cell lines and in peripheral blood lymphocytes (PBL). METHODS: HL-60, K562, DOHH2, Jurkat, MDA435/LCC6, MCF7 cell lines, and PBL derived from normal donors were fixed, permeabilized, stained with anti-Bcl-2-FITC antibody and evaluated by QFCM. In parallel, the same cells were evaluated for Bcl-2 protein expression by Western blot analysis. Mitochondrial localization of anti-Bcl-2-FITC antibody inside cells was confirmed using fluorescence imaging microscopy. RESULTS: Bcl-2 expression in different cell types could be accurately quantified based on antibody-binding capacity (ABC) ranging from 12.6 x 10(3) antibody-binding sites in HL-60 cells to 1.64 x 10(6) antibody-binding sites in a Bcl-2-transfected MDA435/LCC6 clone. The data from flow cytometry analysis correlated well with Western analysis (R(2) = 0.78). Bcl-2-FITC staining colocalized with dyes specific for mitochondria. CONCLUSIONS: The Bcl-2 staining protocol described here was shown to be specific, sensitive, and it was able to provide higher resolution as well as more reproducible quantitation of Bcl-2 protein content in cells when compared with Western blot methods. Quantitation of Bcl-2 content in cells by QFCM may be useful for monitoring Bcl-2 expression in cells undergoing various treatments in vitro and in vivo.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.003 | 0.002 |
| Meta-epidemiology (narrow) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 0.000 |
| Bibliometrics | 0.002 | 0.002 |
| Science and technology studies | 0.000 | 0.001 |
| Scholarly communication | 0.001 | 0.001 |
| Open science | 0.001 | 0.001 |
| Research integrity | 0.001 | 0.002 |
| Insufficient payload (model declined to judge) | 0.003 | 0.002 |
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 itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".