Whole blood fixation and permeabilization protocol with red blood cell lysis for flow cytometry of intracellular phosphorylated epitopes in leukocyte subpopulations
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Notice bibliographique
Résumé
BACKGROUND: Previous studies of intracellular expression of phospho-epitopes in human leukocytes using flow cytometry have used erythrocyte removal or lysis before fixation. Because many of the phospho-epitopes of interest are part of signaling networks that respond to the environment and turn over rapidly, the interval and manipulations used to eliminate erythrocytes from samples have the potential to introduce artifacts. We report a procedure to fix samples containing red blood cells with formaldehyde and then remove erythrocytes by lysis. Detection of phospho-Thr 202/Tyr 204-p44/42 extracellular-regulated kinase (ERK) after phorbol ester acetate (PMA) stimulation was used as a model to measure phospho-epitopes in leukocyte populations in whole blood. METHODS: Normal blood samples were activated with PMA followed by formaldehyde fixation and subsequent treatments with detergents and protein denaturants. The effects of each treatment were monitored by light scatter, selected CD expression intensity, and phosphorylated ERK (pERK) expression. RESULTS: Red cells could be lysed using 0.1% Triton X-100 after brief fixation of whole blood with 2% or 4% formaldehyde. Light scatter improved as a function of formaldehyde concentration and inversely with MeOH concentration. CD3 signal intensity increased when MeOH concentration was reduced. The ratio of pERK immunofluorescence in PMA-stimulated versus nonstimulated (control) samples was highest with high MeOH (90%) and lowest without MeOH treatment. This pattern is consistent with epitope unmasking by alcohol. The pERK epitope could also be unmasked by treatment with high salt, urea, acid, or heat, but none of these produced the level of unmasking of MeOH and each of these was associated with degradation of light scatter and CD3 staining intensity. The final procedure employed 4% formaldehyde, 0.1% Triton X-100, followed by 50% methanol denaturation. Samples prepared in this way demonstrated good preservation of light scatter and surface immunophenotypic patterns, similar to those obtained using a commercial whole blood/red blood cell lysing system (Q-Prep) and an acceptable PMA-stimulated pERK signal (essentially 100% of CD3+ cells that are pERK positive). CONCLUSIONS: Brief fixation of whole blood in 4% formaldehyde followed by treatment with Triton X-100 results in erythrocyte lysis and leukocyte light scatter and immunophenotypic features equivalent to those of other commercial lysis reagents. Intracellular pERK staining is significantly improved by treatment with methanol, but levels of MeOH above 50% degrade light scatter and CD3 expression. This protocol (formaldehyde/Triton X-100/MeOH) circumvents potential artifactual changes in phospho-epitopes due to removal of erythrocytes or erythrocyte lysis followed by fixation, and results in a pERK signal that resolves positive from negative cell populations.
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Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Apprise à partir de 10 348 étiquettes directes de Codex et de 10 348 étiquettes directes de Gemma. Le mode candidate est l'union des têtes enseignantes seuillées; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont ni des étiquettes humaines ni des étiquettes directes de modèles de pointe.
Scores Codex et Gemma par catégorie
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,000 | 0,000 |
| Méta-épidémiologie (sens strict) | 0,000 | 0,000 |
| Méta-épidémiologie (sens large) | 0,000 | 0,000 |
| Bibliométrie | 0,001 | 0,001 |
| Études des sciences et des technologies | 0,000 | 0,000 |
| Communication savante | 0,000 | 0,000 |
| Science ouverte | 0,000 | 0,000 |
| Intégrité de la recherche | 0,000 | 0,000 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,000 | 0,000 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découle