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Adenovirus Titer Protocols
Plaque Assay
Before starting this procedure, prepare 5% agarose as follows:
  (a) Dissolve 2.5 g Agarose in 50ml PBS (pH 7.4).
  (b) Melt in a microwave.
  (c) Store the agarose in sterile, 50ml conical tubes with 5ml per tube.

The viral titer is determined by plaque assay as follows:
1. One day before the assay, plate 293 cells in 6-well plates at a density of ~70%.

2. Serial dilute your virus as follows:
  a. Make a 1:100 dilution by adding 10ul viral stock to 990ul medium.
  b. Starting with a 1:100 dilution, prepare serial 1:10 dilutions by transferring 100ul of diluted virus to 900 l medium up to 10-5 to 10-10 depending on expected concentration of your viral stock.

3. Infect 293 cells with 0.2 ml diluents of adenovirus. Tip the plates to spread the virus evenly over the monolayer.

4. Incubate the cells for 60min at 37°C to allow the virus to infect the cells.

5. During incubation, prepare agarose overlay medium as follows
  a. Melt 5ml of 5% agarose. Then cool to 44°C.
  b. Warm 50ml growth medium to 44°C.
  c. Add 45ml growth medium to 5ml 5% agarose. Mix well.

6. Remove the virus-containing medium from the cells.

7. Gently add 3ml of agarose solution to each well, taking care not to dislodge any cells.

8. When the agarose has set, return plates to incubator. Plaques should be visible within 7C10 days.

9. Prepare a 0.03% solution of neutral red in PBS (1 ml 0.33% [w/v] neutral red stock + 10 ml PBS). Add 1ml of the 0.03% neutral red solution to each of the wells and incubate at 37C for 2C3 hours.

10. Remove the stain by aspiration, then invert the dishes to allow the plaques to clear.
Note : Neutral red is taken up by healthy cells but not by dead cells. Therefore, plaques appear as clear circles against a red or pink background.

11. Calculate Viral Titer by counting the number of well-isolated plaques. Then use the following formula to determine the titer (pfu/ml) of your viral stock:
  #plaques / (D x V) = pfu/ml
  D = dilution factor
  V = volume of diluted virus/well

Sample calculation:
  * An average of 50 plaques formed in the 1:10,000 dilution wells.
  * Volume of diluted virus added: 0.2ml
  50/(0.0001 x 0.2) = 2.5 x 106 pfu/ml
End-Point Dilution Assay
1. A day before the assay, plate 293 cells in two 96-well plates at ~70% density.

2. Prepare serial dilutions of your virus as follows:
  a. Make a 1:100 dilution by adding 10ul virus stock to 990ul medium.
  b. Starting with the 1:100 dilution, prepare serial 1:10 dilutions by transferring 100ul diluted virus to 900ul medium up to 10-3 to 10-10.

3. Add 100ul diluted virus to each well in columns 1-10. Add 100ul of virus-free growth medium to wells in columns 11 and 12. These wells serve as controls for the viability of non-infected cells.

4. Return the plate to incubator for 10 days at 37°C.

5. Using a microscope, check each well for cytopathic effect (CPE). For each column, count the number of wells having CPE. A well is scored as CPE positive even if only a few cells show cytopathic effects.

6. Calculate the fraction of CPE-positive wells in each row. The following shows how you might score a typical End-Point Dilution Assay. In this example, the fraction of CPE-positive wells in each row can be calculated as follows:

7. Calculate Viral Titer:
  Titer (pfu/ml) = 10(1 + Z (X - 0.5))
  Z = Log 10 of the starting dilution (= 1 for a ten-fold dilution)
  X = the sum of the fractions of CPE-positive wells
Note: Even if you omit some of the lower dilutions (e.g. the 1:10 and 1:100 dilutions) as in the example above, you must count them as part of the sum. The formula given is based on the Spearman-Karber method.

Sample calculation:
X = (1 + 1 + 1 + 1 + 1 + 0.7 + 0.4 + 0.2 + 0 + 0) = 6.3
Titer = 10(1 + 1 (6.3 - 0.5)) = 10(1 + 1 (5.8)) = 10(1 + 5.8) = 106.8
Titer = 106.8 = 6.3 x 106pfu/ml

The assay is a reliable indicator of viral titer only if the following three conditions are met:
  * The negative control wells are free of CPE.
  * Wells infected with the least dilute virus (10-3 in the example) are all CPE-positive.
  * Wells infected with the most dilute virus (10-10 in the example) are all CPE-negative.
OD260 Assay
This assay is used for determining the titer of concentrated stocks of purified adenovirus. It should not be used for measuring virus in crude cell lysates or in culture supernatant since serum and other factors in growth media interfere with the absorbance at 260 nm.

1. Purify and concentrate recombinant adenovirus by banding in a CsCl density-gradient.

2. Prepare dilutions of your virus as follows:
Dilution Virus stock 0.1% SDS Buffer*
1:10 50l 450l
1:25 20l 480l
1:50 10l 490l
* Prepare 0.1% SDS as a solution in any suitable buffer (e.g., 1X TE or PBS).
In general, appropriate dilutions for assay are those having an OD260 in the range 0.1C1.0. Remember to make 0.5-ml blanks for each dilution by substituting TE buffer (or equivalent dialysis buffer) for your virus stock.

3. Record the absorbance at 260 nm (OD260). Always start by reading the absorbance of the most dilute sample:
  * Fill the cuvette with the blank for the 1:50 dilution.
  * Record the OD260.
  * Discard the blank.
  * Add the 1:50 dilution and read its absorbance.
  * Rinse the cuvette once before adding the blank for the next dilutionthe 1:25 dilution.
  * Repeat Steps aCe for the 1:25 and 1:10 dilutions.

4. Record the absorbance at 280 nm (OD260).

5. Calculate viral titer and purity:
  viral titer (opu/ml) = OD260 x viral dilution x 1.1 x 1012
  opu = optical particle unit
Note: Because optical particle units (opu) and plaque forming units (pfu) define different properties, these measurements cannot be directly compared. Purity = OD260/OD280, typically ~1.2-1.3 after CsCl purification.