A549-ACE2 cell line ACE2新冠靶点稳定表达人肺腺癌细胞株 BioVector NTCC质粒载体菌种细胞基因保藏中心

A549-ACE2 cell line ACE2新冠靶点稳定表达人肺腺癌细胞株

CELL LINE DESCRIPTION
A549-hACE2 cells were generated from the A549 lung carcinoma
cell line, a commonly used cellular model for the study of respiratory
infections. A549-hACE2 cells were stably transfected to express the
human ACE2 (hACE2) gene. Thus, unlike their parental cell line, they
are permissive to infection with pseudotyped lentiviruses expressing
the SARS-CoV-2 Spike protein.
. A549-hACE2 cells are resistant to
Puromycin.
The additional expression of the human TMPRSS2 gene in the A549-
hACE2-TMPRSS2 cells significantly increases their permissivity to
infection by SARS-CoV-2 Spike-pseudotyped lentiviruses.

BACKGROUND
ACE2 (angiotensin I-converting enzyme-2) is a type I membrane
protein that belongs to the angiotensin-converting enzyme family1
. It
is expressed in arteries, heart, kidneys, and epithelia of the lung and
small intestine2
. Human ACE2 is the established host receptor for
the Spike (S) protein of SARS-CoV-2, the causative agent of COVID19, enabling its entry into target cells3-5. In particular, SARS-CoV-2
gains entry to host cells through the binding of the Spike receptorbinding domain (RBD) to ACE2 at the cell surface4,5. Following
this, host proteases, such as TMPRSS2 and Cathepsin L, allow the
cleavage of the S protein into two subunits (S1 and S2), at the cell
surface or in the endosomes, respectively. S2 mediates the fusion
between the viral and host membranes, thereby releasing the viral
contents into the cell.

APPLICATIONS
A549-hACE2 cells are permissive to infection by SARS-CoV-2 and/or
spike-pseudotyped lentiviral particles. Thus, they are ideal for studying
viral entry into host cells, as well as for screening small molecule
inhibitors and neutralizing antibodies. These cells can be used for
comparative studies with A549-hACE2-TMPRSS2 cells which express
both ACE2 and TMPRSS2 and are more permissive to SARS-CoV-2
infection than A549-hACE2.

HANDLING PROCEDURES
Required Cell Culture Medium
• Growth Medium: DMEM, 4.5 g/l glucose, 2 mM L-glutamine,
10% heat-inactivated fetal bovine serum (FBS; 30 min at 56 °C),
100 μg/ml Normocin™, Pen-Strep (100 U/ml-100 µg/ml)
• Freezing Medium: DMEM, 4.5 g/l glucose, 10% FBS, 10% DMSO
• Required Selection Antibiotic: Puromycin
Initial Culture Procedure
The first propagation of cells should be for generating stocks for
future use. This ensures the stability and performance of the cells for
subsequent experiments.
1. Thaw the vial by gentle agitation in a 37 °C water bath. To reduce
the possibility of contamination, keep the O-ring and cap out of the
water. Thawing should be rapid (approximately 2 minutes).
2. Remove the vial from the water bath as soon as the contents are
thawed, and decontaminate by dipping in or spraying with 70% ethanol.
Note: All of the steps from this point should be carried out under strict
aseptic conditions.
3. Transfer cells to a larger tube containing 15 ml of pre-warmed
growth medium. Do not add selection antibiotics until the cells
have been passaged twice.
4. Centrifuge tube at 200-300 x g for 5 minutes.
5. Remove supernatant containing the cryoprotective agent and
resuspend cells with 1 ml of growth medium without selective
antibiotics.
6. Transfer the contents to a T-25 tissue culture flask containing 5 ml
of growth medium without selective antibiotics.
7. Place the culture at 37°C in 5% CO2
.
Frozen Stock Preparation
1. Resuspend cells at a density of 5-7x 106 cells/ml in freshly
prepared freezing medium.
Note: A T-75 culture flask typically yields enough cells for preparing 1-2
frozen vials.
2. Dispense 1 ml of cell suspension into cryogenic vials.
3. Place vials in a freezing container and store at -80°C overnight.
4. Transfer vials to liquid nitrogen for long-term storage.
Note: If properly stored, cells should remain stable for years.
Cell maintenance
1. A549-hACE2 cells grow as adherent cells. To detach cells, rinse
the cell layer with PBS, then incubate with 0.25% trypsin-EDTA for
2-5 minutes.
2. After cells have recovered and are growing well (following at least
2 passages), maintain and subculture the cells in growth medium
supplemented with 0.5 μg/ml of Puromycin.
3. Renew growth medium twice a week.
4. Cells should be passaged when a 70-80% confluency is reached. Do
not let the cells grow to 100% confluency.
Note: The average doubling time for the A549-hACE2 cells is ~25 hours
using the conditions described above.
Cell Handling Recommendations
To ensure the best results, use A549-hACE2 cells with less than 20
passages.



Supplier来源:BioVector NTCC Inc.
TEL电话:400-800-2947
Website网址: http://www.biovector.net