Abstract
O vírus sincicial respiratório humano (HRSV) infecções apresentar um largo espectro de gravidade da doença, variando desde as infecções suaves a bronquiolite com risco de vida. Uma parte importante da patogênese da doença grave é uma resposta imune melhorada levando a imunopatologia.
Aqui, nós descrevemos um protocolo usado para investigar a resposta imune de células do sistema imunológico humano a uma infecção HRSV. Em primeiro lugar, descrevemos métodos utilizados para a cultura, purificação e quantificação de HRSV. Subsequentemente, foi descrito um modelo humano in vitro, em que as células mononucleares do sangue periférico (PBMC) são estimuladas com HRSV vivo. Este sistema modelo pode ser usado para estudar vários parâmetros que podem contribuir para a gravidade da doença, incluindo a resposta imune inata e adaptativa. Estas respostas podem ser medido ao nível da transcrição e da tradução. Além disso, a infecção virai de células pode ser facilmente medida utilizando citometria de fluxo. Tomadojuntos, a estimulação de PBMC com HRSV vivo fornece um sistema modelo rápido e reprodutível para examinar os mecanismos envolvidos na doença induzida por HRSV.
Materials
Name | Company | Catalog Number | Comments |
Reagent | |||
PBS | Lonza | BE17-516F | PBS without Ca2+ Mg2+ or phenol red |
HBSS | Invitrogen | 14025-100 | HBSS, Calcium, Magnesium, no Phenol Red |
DMEM | Invitrogen | 31966-047 | DMEM, High Glucose, GlutaMAX, Pyruvate |
RPMI | Invitrogen | 72400054 | RPMI 1640 Medium, GlutaMAX, HEPES |
Reduced Serum Medium (Opti-MEM) | Invitrogen | 51985-026 | Opti-MEM I Reduced Serum Medium, GlutaMAX |
FCS | Greiner Bio-one | FBS (Fetal Bovine Serum) | |
BSA | Sigma Aldrich | A7030 | Albumin from bovine serum |
P/S | Invitrogen | 15140-122 | Penicillin-Streptomycin, liquid |
Trypsin | Invitrogen | 25300-054 | 0.05% Trypsin-EDTA (1x), Phenol Red |
HeLa cells | ATCC | CCL-2 | |
A549 cells | ATCC | CCL-185 | |
Sucrose | Sigma Aldrich | S7903 | Sucrose BioXtra, ≥99.5% |
IgG2 anti-mouse-FITC | BD Pharmingen | 555057 | Ms IgG2b K FITC isotype control |
Anti-NP-RSV-FITC | Abcam | ab25849 | Anti-Respiratory Syncytial Virus antibody [671] (FITC) |
Density gradient medium (Lymphoprep) | Axis-Shield | 1114545 | |
EDTA tubes | BD Biosciences | 367525 | |
Acetone | Merck Millipore | 1000141000 | Acetone for analysis |
Material | |||
Centrifuge Tubes | Beckman Coulter | 326823 | Thinwall, Polyallomer, 38.5 ml, 25 mm x 89 mm |
SureSpin 630 Rotor with 36 ml buckets | Sorvall | 79368 | Swinging bucket titanium rotor |
Sorvall WX80 Ultracentrifuge | Sorvall | 46900 | |
CB 150l CO2 Incubator | Binder |
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