Measurement of nitric oxide in murine Hepatoma Hepa1c1c7 cells by reversed phase HPLC with fluorescence detection.
Bibliographic record
Abstract
PURPOSE: Nitric oxide (NO) is produced by various cell types in picomolar to nanomolar range and has important roles in a variety of biological functions. The aim of the present study was to investigate a sensitive and reproducible fluorometric HPLC method for measuring nitrite, one of the stable oxidation products of nitric oxide, in murine hepatoma Hepa 1c1c7 cells. METHODS: Hepa 1c1c7 cells were incubated with vehicle or recombinant murine tumor necrosis factor-alpha (TNF-alpha, 10 ng/ml) in Hanks' balanced salt solution (HBSS) for 10 hours. Thereafter, the HBSS medium was collected for nitrite analysis. NO production was examined by measuring the conversion of 2, 3-diaminonaphthalene (DAN) to its fluorescent product, 2, 3-naphthotriazole (NAT). NAT was analyzed after elution with 60% of 15 mM sodium phosphate buffer (pH = 7.5) and 40% methanol through a 10-microm reversed-phase C18 column (250 x 4.00 mm, I.D.) at a flow rate of 1 ml /min. Fluorescence was monitored with excitation at 375 nm and emission at 415 nm. RESULTS: NAT appeared in approximately 16 min with no interference peaks. The assay yielded linear response within the examined range of 10 - 200 pM (r(2) >0.99) with an intra and inter-day variability of <10 % and accuracy of > 90%. The detection limit for nitrite was 10 pM. NO production by TNF- alpha treated Hepa 1c1c7 cells is estimated to be approximately 2 folds more than untreated cells. CONCLUSION: This fluorometric HPLC assay offers a sensitive and reliable measurement of NO production in cell culture medium.
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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 | 0.001 | 0.000 |
| Meta-epidemiology (narrow) | 0.001 | 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 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".