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Apr 03, 2019

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# DENV Titration

DOI

https://dx.doi.org/10.17504/protocols.io.zsdf6a6

Fabio Gomes¹

¹Johns Hopkins University

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Fabio Gomes

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DOI: https://dx.doi.org/10.17504/protocols.io.zsdf6a6

Protocol Citation: Fabio Gomes 2019. DENV Titration. protocols.io https://dx.doi.org/10.17504/protocols.io.zsdf6a6

License: This is an open access protocol distributed under the terms of the **Creative Commons Attribution License**, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Protocol status: Working

We use this protocol and it's working

Created: April 03, 2019

Last Modified: April 03, 2019
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Protocol Integer ID: 22053

Troubleshooting
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## Fixation of Cells

1. Prepare fixative - 80% methanol in water

2. Dump Methylcellulose overlay and blot on paper towels

3. Wash gently with 1X PBS. Incubate for 10 min. Blot and dry. 00:10:00

4. Add 0.5 mL fixative to each well. Allow plates to sit at room temperature for 10 min.

**Note**

Note: Fixed cells can be stored at -70°C for future use. Leave methanol on cells when freezing.

## Antibody

5. Wash plates with PBS. Incubate for 10 min. 00:10:00

**Note**

If frozen plates are being used, incubate at 37°C for 30 min.

6. Prepare antibody dilutions in 5% skim milk, PBS [M] 5 Mass / % volume Skim Milk

**Note**

4G2 1:2000
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7 Add 200 µl to each well. Incubate at 37oC for 1 hour on a rocker. 01:00:00

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37 °C

Minimize contact with each well by maintaining contact of the pipette tip with the wall of well.

8 Dump off primary antibody solution and tap plates on paper towels to remove excess solution

9 Wash with 1 mL 5% skim milk, PBS

10 Prepare antibody dilutions in 5% skim milk, PBS.

Note

Anti-mouse-Per 1:2,000

11 Add 200 µl to each well. Incubate at 37oC for 1 hour on a rocker. 01:00:00

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37 °C

Color Development

12 Dump off antibody solution. Tap on paper towels

13 Wash twice with PBS. Tap on paper towel

14 Add 160 µl of TrueBlue substrate per well

15 Place on rocker at RT until plaques develop (10 min or longer).
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16 Dump off peroxidase substrate.

17 Count plates or incubate at 4oC for up to 7 days.