gap sterile electroporation cuvette 1652081  (Bio-Rad)

Journal: Methods and Protocols

Article Title: Formalin and 2.5% Glutaraldehyde/2% Paraformaldehyde in 0.1 M Cacodylate Buffer Inactivation Protocols to Ensure the Proper Fixation of Positive Sense RNA Viruses and Genomic Material Prior to Removal from Containment

doi: 10.3390/mps7060105

Figure Lengend Snippet: Mechanisms of introduction of positive-sense viral RNA into cells. ( A ) Depicts infection of a host cell with a natural EEEV particle. The virus entry protein, E2, binds to a host receptor (1) that allows for entry into the host cell via endocytosis (2). Within the endosome, the pH decreases, allowing for conformational changes of the entry and fusion proteins, enabling the fusion protein, E1, to create a pore within the endosome, resulting in the release of the viral capsid into the cytosol (3). The capsid is then disassembled by the host ribosome, and viral genomic RNA is released, transcribed, and translated into viral proteins (4). Structural proteins and genomic RNA then assemble and bud at the peripheral membrane to form progeny virions. ( B ) Depicts intentional introduction of the viral genomic RNA via electroporation for comparison. (1) Electroporation creates pores in the peripheral membrane, (2) allowing artificially introduced EEEV genomic RNA to enter the cell without the viral structural proteins typically needed for entry and fusion steps. (3) Replication, transcription, and translation then occur in the same manner as natural virus infection, (4) producing progeny virions that can infect and replicate in the same manner as 1A.

Article Snippet: Up to 200 μL of RNA from inactivated (formalin or glut/PFA) or control samples was added to a 4 cm gap sterile electroporation cuvette (BioRad, 1652088).

Techniques: Infection, Virus, Membrane, Electroporation, Comparison

Journal: Methods and Protocols

Article Title: Formalin and 2.5% Glutaraldehyde/2% Paraformaldehyde in 0.1 M Cacodylate Buffer Inactivation Protocols to Ensure the Proper Fixation of Positive Sense RNA Viruses and Genomic Material Prior to Removal from Containment

doi: 10.3390/mps7060105

Figure Lengend Snippet: EEEV-infected NHP brains were fixed in 10% formalin and homogenized alongside positive control tissue samples. RNA extraction was then performed on samples. RNA was then electroporated into BHK-21 cells. RNA samples that were more than 0.5 (electroporation volume) were split into separate samples (ex: 3-1 into 3-1-1, 3-1-2, and 3-1-3) for electroporation into cells. Cells were then placed in flasks for 72 h as a first passage. A second passage was also performed for 72 h. CPE was observed and recorded daily. PFU controls were created by adding diluted stock virus at the designated PFU directly to cell culture. Plaque assay was performed on RNA extracted from formalin-fixed samples and control samples to determine if genomic replication and infectious virus production were occurring. Stock virus was diluted and used as a plaque assay positive control. The limit of detection for the assay was 1 PFU/mL. ( A ) CPE and plaque assay data for replicate experiment 1. ( B ) CPE and plaque assay data for replicate experiment 2. ( C ) CPE and plaque assay data for replicate experiment 3.

Article Snippet: Up to 200 μL of RNA from inactivated (formalin or glut/PFA) or control samples was added to a 4 cm gap sterile electroporation cuvette (BioRad, 1652088).

Techniques: Infection, Positive Control, RNA Extraction, Electroporation, Virus, Cell Culture, Plaque Assay, Control