TITLE: SARS-CoV-2 TCID50
AUTHORS: Briana L McGovern

[DESCRIPTION]
Protocol used to titer both animal tissue samples and viral stocks at BSL-3

[STEPS]
SECTION: Seeding
2. The day before the assay, seed Vero-TMPRSS2 cells in 96-well plates @ 2e4 cells/well (2e5 cells/ml).
You can either perform the TCID50 in triplicate, which would have 4 samples per plate
or 4x, which would have 3 samples per plate. 

Always check the confluency of the cells on the day of the assay. If the seeding was not proper you will not get valid results on the TCID50.
SECTION: Deep Well Prep
3. The day before the assay, fill the   with 900ul 2% media. Then label the side of the deep well with the samples IDs. Each deep well can do 12 samples.
Cover the plates with   and store at 4 °C until the assay.

You should also label the plates with matching sample IDs and make sure to add numbers to the side of the lid and plate to avoid any mix up of IDs later on.

 

 

SECTION: Media Prep
1. Make 10% media (Vero Cell Growth Media):
500ml  
5ml  
5ml   
50ml FBS
150ul Puromycin
1ml plasmocin

Make 2% media (Infection Media):
500ml   
5ml   
5ml   
10ml FBS

SECTION: Prepping for BSL-3 TCID50
4. Pack:
2 sleeves per person
1 mask per person
1 gown per person
1 hairnet per person
2 shoe covers per person
small and medium biohazard bags
the samples (if not already in the facility)
the plates
the deep well plates
200ul filtered tips - each box can do 3 or 4 plates depending on the amount of replicates you chose in step 2.
SECTION: Setting up for the assay in the BSL-3
5. Once you've donned the PPE, entered the facility, entered your booked BSC and set up the coffin, you can set up for ideal TCID50 work flow -
IMMEDIATELY take out your samples so that they begin thawing.
Store the plates in the incubator since there is a good amount of work done before plating.
SECTION: Setting up for the assay in the BSL-3
5.1. Sample Prep:
Once the samples are thawed, move the centrifuge into the BSC. You cannot operate the centrifuge on the bench!
Centrifuge the samples for 5 minutes at 5,000rpm to aggregate the tissue at the bottom of the tubes. You will titer the supernatant.

SECTION: Setting up for the assay in the BSL-3
5.2. While the samples are spinning you can set up the BSC. 

When working with >20 samples it is best to work in a pair! One person will remove media from the plates while the other person treats the plates with the dilutions.

Have 2 boxes of tips on each side of the BSC.
Split the deep wells into 2 groups so that you and your partner can fill deep wells with samples at the same time.
SECTION: The TCID50
6. Once the samples have been spun down, organize them to match the order of the deep wells.

Add 100ul supernatant to the first row of the deep wells. Replace the parafilm.

Once all the deep wells have been loaded you will proceed one deep well at a time.
SECTION: The TCID50
6.1. Perform the serial dilution for the first deep well:
Using a multichannel pipette mix the first row ~20 times.
Move 100ul of the first row to the following row, mix ~15 times.
Repeat until the entire deep well has been done and all the dilutions have been performed.
SECTION: The TCID50
6.2. Remove the plates from the incubator that match the deep well.
One person should remove the media from the entire plate and slide it to the next person.
That person will then move 200ul of the dilution series to the plates in the amount of replicates that you prepared for.

I recommend having the deep well, the plate, and the tip box in a vertical format. You can then easily move the dilutions in the proper order to the plates.

 

SECTION: The TCID50
6.3. Incubate until CPE is visible.
Typically 3 days for in vivo lung titer analysis.

TCID50 is also used for titering viral stocks, follow the same protocol without sample centrifugation.
3 days for SARS-CoV-2 WA1 and early variants (alpha, beta, mu, etc)
4 days for Omicron
3-4 days for later variants (ex XBBs, JN.1, BAs, etc) The incubator for these later variants varies, you should check the CPE at day 3 and decide.
SECTION: Fixing
7. After 3 days you will return to the facility with your PPE and a reservoir.
Add 100ul 10% PFA to all wells on the plates.
Gently spray the lids with ethanol and replace.
Double bag the plates in red biohazard bags and bring down to the lab. Do not handle the plates for 1 day.
SECTION: Staining
8. Remove the plates from the bags and bring them to the fume hood. Remove the liquid from the wells and discard in the formaldehyde waste container.

Add 75ul 1% crystal violet and incubate for 15 minutes.

Remove the crystal violet and discard in the appropriate waste container.

Bring the plates back to the lab and wash them well using tap water to reveal CPE.

Leave the plates with lids open to dry

SECTION: Counting CPE
9. Once the plates have dried a bit you can take a look at the CPE and count.

Replace the lid and flip the plate over to visualize staining. You may have to wipe dry to write on the plate. Divide the plate by the samples and count the amount of CPE positive wells in each row. Record these numbers (or take photos) for analysis.

 

SECTION: CPE Analysis
10. Alter the conditions of the excel sheet to match what you had performed for this experiment.
Plug in the positive and negative CPE values into the excel sheet.
The sheet will spit out a TCID50/ml value. Record these values