MDCK cell separation method for influenza viruses

, purpose
MDCK cell separation method of influenza virus is the key technology of influenza experiment, which is used for the separation,
culture
. This SOP is designed to ensure the accuracy, specification and reliability of the operation of MDCK cells to isolate influenza viruses.
, the range
suitable for all laboratory technicians to carry out MDCK cell separation of influenza viruses.
III, Procedure
(i) Biosecurity Requirements
H5, H7 subtype highly pathogenic avian influenza virus, H2N2 subtype influenza virus MDCK cell isolation laboratory biosecurity level: BSL-3. The experimental operator is required to carry out BSL-3 protection, as detailed in
Biosecurity
SOP. H5, H7 subtype of highly pathogenic avian influenza virus, H2N2 subtype influenza virus MDCK cell separation operation must be carried out in the BSL-3 level laboratory
biosecurity cabinet
.
level of MDCK cell isolation laboratory biosecurity for the remaining influenza viruses: BSL-2. The experimental operator is required to carry out BSL-2 protection, as detailed in biosecurity personal protection SOP. The MDCK cell separation of the remaining influenza viruses must be performed in the biosecurity cabinet of the BSL-2 laboratory.
(ii) material
1, 75% to 90% of the pieces of MDCK cells, choose a suitable size
cell culture
bottles and cell culture plate to do virus isolation
2, pancrease (cow pancreas source VIII.type)

3, HEPES buffer, 1M mother liquid
4, D-MEM
culture base
, Hank's liquid
5, blue, streptomycin master solution (10000U/mL penicillin G;1 10000 mg/mL streptomycin
6, bovine
serum
albumin part V., 7.5% solution
7, clinical sample 0.5mL
8, 1mL sterile pipet
9, 10mL sterile pipe
10, 15mL sterile
centrifugal tube
(iii) experimental step
1, preparation of viral growth fluid
(1) cell maintenance fluid preparation

(1) blue, streptomycin mother solution 5mL (end concentration up to: 100U/mL penicillin; 100m/mL streptomycin)
(2) bovine serum albumin V.12.5mL (final concentration: 0.2%) in 500mL D-MEM
(3) HEPES buffer 12.5mL (final concentration: 25mM)
(2) viral growth fluid
adds 0.5mL of TPCK-pancrease (mother fluid concentration of 2mg/mL) to the 500mL cell maintenance fluid to bring the final concentration of TPCK-pancrease to 2μg/mL.
2． Influenza virus MDCK cell separation steps:
(1) 75% to 90% of the preparation of tablet cells, to choose T25 cell bottles as an example.
(1) look at cell growth × 40-minute objector.
(2) gently pour out the cell growth fluid, using a 10mL sterile pipet to absorb 6mL Hank's liquid to clean the cells 3 times.
(2) Cell
Culture Bottle
Inoculation
(1) Remove the Hank's liquid cleaning cell from the cell culture bottle using a sterile pipet.
(2) the appropriate amount of clinical specimens were absorbed from sterile pipelets and placed in cell culture bottles, shaking gently several times.
(3) and then put in 37 degrees C, 5% CO2 culture tank adsorption 1 to 2h.
(4) to suck out the inoculation, using a 10mL sterile pipet to absorb 6mL Hank's liquid to clean the cells 2 times. The 6mL virus growth fluid is then added to the cell culture bottle.
(5) is placed in a culture box culture of 33 to 35 degrees C.
(6) daily observation of cell lesions. (Cell lesions are characterized by cell swelling rounding, increased cell gap, cell nucleation or rupture, and partial or total cell shedding in severe cases.)
(3) Harvest of cell culture
when 75% to 100% of cells become lesions, before harvesting can be put in the refrigerator at -70 degrees C, frozen and melted 1 to 2 times, to improve the viral titration of the harvest specimen. Even cell-free lesions should be harvested on the 7th day after vaccination. When harvesting the viral fluid, gently shake the cell bottle several times, and then use a 10mL sterile pipe to absorb the virus liquid placed in a 15mL sterile centrifuge tube, mixing the virus. The harvested viral fluid can be tested immediately or frozen in a refrigerator at -80 degrees C for later testing.