The applied assay was established previously by Stauffer et al. (23 (link)). Prior to infection, cells were preincubated with bafilomycin A1 (Cayman catalog no. cay-11038) (250 nM). Virus (PR8M) was primed for 1 h at 37°C with DMEM adjusted to pH 5.8 with MES (morpholineethanesulfonic acid) (30 mM). Cells were infected at an MOI of 50 with primed virus for 1 h on ice and subsequently washed twice with cold DMEM-BA. An acidic bypass was achieved by incubation with low-pH DMEM (50 mM citrate, pH 5.0) for 2 min, followed by two washing steps with cold DMEM-BA. DMEM-BA containing bafilomycin A1 (250 nM) was added, and cells were incubated for 8 h at 37°C. For fluorescence-activated cell sorter (FACS) analysis, the cells were harvested by Accutase treatment (Millipore catalog no. SCR005), fixed in 4% formaldehyde for 10 min, and permeabilized by the use of 0.1% Triton X-100 for 30 min. Blocking as well as staining with an anti-NP antibody (mouse anti-influenza A virus nucleoprotein, clone AA5H; AbD Serotec [MCA400]) for 2 h and anti-mouse Alexa Fluor 488 (donkey anti-mouse IgG secondary antibody) (Alexa Fluor 488; Invitrogen catalog no. R37114) for 1 h was done using 2% BSA–PBS++. Cells were washed three times between blocking and antibody incubations. A total of 10,000 cells were analyzed per sample. Uninfected cells or cells without bypass served as a control.
Mca400
Manufactured by Bio-Rad
Sourced in Germany, United States
The MCA400 is a compact and versatile microplate centrifuge from Bio-Rad. It is designed to quickly spin down microplates, PCR tubes, and other small sample containers. The MCA400 features a brushless motor and a robust, stainless-steel construction for reliable performance and long-term durability.
Automatically generated - may contain errors
Lab products found in correlation
Avidin biotin detection system, by Roche (2 mentions)
Redmap kit, by Roche (2 mentions)
H1009, by Merck Group (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Lsm 800 microscope, by Zeiss (1 mentions)
Plan apochromat 63 1.4 oil immersion objective, by Zeiss (1 mentions)
Zoe fluorescent cell imager, by Bio-Rad (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Gtx125928, by GeneTex (1 mentions)
Anti rabbit secondary antibody conjugated to alexa 594, by Thermo Fisher Scientific (1 mentions)
Anti mouse secondary antibody conjugated to alexa 488, by Thermo Fisher Scientific (1 mentions)
Avicel ph 101, by Merck Group (1 mentions)
8 protocols using mca400
1
Influenza Virus Entry Inhibition Assay
2
Influenza Virus Antigen Distribution
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Paraffin-embedded sections were cut (5 μm), dewaxed, and stained with hematoxylin and eosin. Duplicate sections were immunohistochemically analyzed to determine the distribution of influenza virus antigens in individual tissues. Briefly, sections were stained with a mouse monoclonal antibody against influenza A virus nucleoprotein (MCA-400; AbD Serotec, Duesseldorf, Germany), followed by a biotinylated goat anti-mouse IgG secondary antibody. Bound antibodies were detected with an avidin-biotin detection system (Ventana Medical Systems, Tucson, AZ, USA). The RedMap kit (Ventana Medical Systems) served as the substrate chromogen.
3
Histopathological Analysis of Influenza Infection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
On day 4 p.i., parenchymal tissues were collected for histopathological analysis. Collected tissues were fixed for 24 h in 10% buffered neutral formaldehyde and then were processed for paraffin embedding. Paraffin sections were cut into 5-µm-thick sections, dewaxed, and then stained with hematoxylin and eosin. Duplicate sections were used in immunohistochemical analyses to assess the distribution of influenza viral antigens in individual tissues. Briefly, sections were stained with a mouse monoclonal antibody against influenza A virus nucleoprotein (MCA-400, AbD Serotec, Duesseldorf, Germany) and then were treated with a biotinylated goat anti-mouse-IgG secondary antibody. Bound antibodies were detected with an avidin-biotin detection system (Ventana Medical Systems, Tucson, AZ, USA). The chromogenic substrate was provided by the RedMap kit (Ventana Medical Systems).
4
Microneutralization Assay for Antibody Titer
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The microneutralization (MN) assay was used for measuring neutralizing antibodies as previously described (13 (link)). Sera were heat-inactivated at 56°C for 30 minutes and added in 3-fold serial dilutions to a ninety-six-well cell culture plate (Nunclone Delta surface, USA), with H1N1pdm09 like-virus (X179a, 2000 TCID50/ml). Madin-Darby Canine Kidney (MDCK) cells were added after one hour incubation in room temperature, and the plates were then incubated in 37°C for 18 hours. The next day, cells were fixed with hydrogenperoxide (Sigma-Aldrich, USA, H-1009) in methanol (Sigma-Aldrich, USA, 32213) (0.6% H2O2) in 20 minutes. Mouse anti-influenza A nucleoprotein antibodies (AbD Serotec, USA, MCA400) was added, and the plates were incubated for one hour at 37°C. HRP secondary antibody (Dako, Denmark, P0260) was added followed by one-hour incubation. Influenza virus was detected using TMB, before reading at 450 and 620nm to calculate the OD-value. The dilution of serum that provided 50% inhibition of infection was calculated as the MN titre. Negative values were assigned a value of 5 for calculation purposes.
5
Influenza Virus Nucleoprotein Immunostaining
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were pretreated as indicated in the respective figure legends, seeded on covers slips 1 day prior to infection, and infected with PR8M by a standard procedure as described above. At 4 h p.i., cells were fixed (4% PFA–PBS++ for 10 min), permeabilized (0.1% Triton X-100–PBS++ for 30 min), and blocked (2% BSA–PBS++, overnight, 4°C). Staining with anti-NP antibody (mouse anti-influenza A virus nucleoprotein, clone AA5H; AbD Serotec [MCA400]) for 2 h and anti-mouse Alexa Fluor 594 (donkey anti-mouse IgG [H+L] secondary antibody, Alexa Fluor 594; Invitrogen catalog no. A21203) for 1 h with DAPI (4′,6-diamidino-2-phenylindole) (Sigma) was done using 2% BSA. Transfected cells were identified by confocal microscopy using an LSM 800 microscope (Carl Zeiss, Inc., Jena, Germany) equipped with a Plan-Apochromat 63×/1.4 oil immersion objective and were categorized for the presence or absence of distinct IAV NP signal.
6
Quantifying Viral Antigen in Infected Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To analyze primary infection, A549, and HEL cells as well as hAECB were washed with PBS and incubated for 2 h with small molecules (3 μM) or an equivalent of DMSO under normal culture conditions. Then, cells were inoculated with an MOI of 0.1 (CPXV, HSV1, VSV), 0.3 (IVs), or 1.5 (HTV), respectively, for 30 min at 4°C. Unbound virus was removed and the cells cultivated for 6 h (IV, VSV) or 24 h (CPXV, HSV1, HTV) in presence of small molecules. Then, the rate of infected cells was determined by microscopically quantifying the number of cells positive for viral antigen. In case of IVs and VSV, presence of the NP protein was investigated by indirect immunofluorescence analysis using primary antibodies MCA400 (IAVs, AbD Serotec), MCA403 (IBVs, AbD Serotec), polyclonal-anti hantavirus N protein and V5507 (VSV, Sigma-Aldrich). In case of CPXV and HSV1, presence of GFP was investigated by fluorescence analysis. Microscopic analysis was performed as described previously [11 (link)].
7
Influenza A Virus NP and M1 Immunostaining
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Influenza A/WSN/33 (H1N1) infected cells were fixed with 4% formaldehyde for 10 min followed by permeabilization with 0.2% Triton X-100 for another 10 min. After blocking with 10% bovine serum, cells were stained with mouse anti-NP (1:200 dilution; Bio-Rad: MCA400, Hercules, CA, USA) and rabbit anti-M1 (1:200 dilution; Gene Tex: GTX125928) antibodies, followed by incubation with anti-mouse secondary antibody conjugated to Alexa-488 and anti-rabbit secondary antibody conjugated to Alexa-594 (Thermo Fisher Scientific, Waltham, MA, USA). Nuclei were stained with 300 nM DAPI (Thermo Scientific) after secondary antibody incubation. Fluorescent images were acquired using a ZOE Fluorescent Cell Imager (Bio-Rad).
8
Virus Purification and Titration in MDCK II Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Virus purification and virus titration was performed in MDCK II cells as described before [14 (link)]. The viral titer was determined by plaque assay as previously described [15 (link)]. Briefly, confluent MDCK II cells were infected with serially diluted virus or tissue supernatant (in MDCK II culture medium without FCS). Virus was allowed to adsorb to the cells for 1 h at 37°C. Then, the inoculum was replaced with fresh media (supplemented with 1 μg/mL TPCK treated trypsin (for IAV) and 1% Avicel® PH-101 (Sigma-Aldrich)). MDCK II cells were stained with crystal violet or virus-infected cells were stained with primary antibody (1:2,000) (mouse anti-influenza A nucleoprotein, MCA400, Bio-Rad, Germany) and the secondary antibody (1:4,000) (Goat Anti-Mouse IgG-HRP, 170–6516, Bio-Rad, Germany) for plaque assay. The plaques were manually counted and expressed as plaque forming units (pfu) per mL.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!