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This protocol is for the stable cell line construction based on puromycin selection.

Day 1: Seed target cells in 24-well plates. The number of seeding cells differs according to the cell proliferation rate.
Day 2: Target cells should be approximately 50%-70% confluent. For polybrene accessible cells, mix the culture medium with proper concentrations of polybrene. Replace the medium completely with 0.5 ml polybrene-containing medium. For polybrene sensitive cells, this step can be skipped.
Before infection, virus should be melted on ice gently and resuspended in culture medium. Remove the preceding medium and add lentivirus-containing medium with 1/2 volume of normal culture volume. Culture for 4 hours at 37℃, and supplement fresh medium to normal volume. The recommended medium volume of lentivirus infection is displayed in the following table 4.

Table 4. Medium volume of lentivirus infection (1/2 volume for lentivirus infection)
Culture dish type Surface area normal volume for cell culture 1/2 volume for lentivirus infection
96-well 0.3 cm2 100 ul 50 ul
24-well 2 cm2 500 ul 250 ul
12-well 4 cm2 1 ml 500 ul
6-well 10 cm2 2 ml 1 ml

1. Polybrene concentration
Though polybrene increases the efficiency of viral infection, it is toxic to some cell lines, and the sensitivity varies from different cell lines. For polybrene accessible cells We recommend the working concentration of polybrene as 6-8μg/ml.
2. Optimal MOI detection for cell infection
MOI (multiplicity of infection) refers to the number of infected viral particles per cell. For actively dividing cells such as HeLa or 293 cells, over 80% of the cells can express target genes with MOI of 1-3. For the non-dividing cells like primary cells with a low infection efficiency, we recommend testing a range of MOIs to determine the optimal MOI for infection and gene expression in target cell lines. The lentiviral MOIs of some common cell line are shown in the table 5.

Table 5. The lentiviral MOIs of common cell line.
Cell line MOI range Auxiliary infection reagent polybrene (need/no)
K562 20~40 Need
Jurkat 50~80 no
kasumi 10~30 no
NB4 50~80 no
U937 20~40 Need
THP-1 50~80 Need
GBC-SD 30~50 no
H929 100~150 no
H1299 1~3 Need
95D 2~4 Need
A549 20~40 Need
SPC-A-1 100~150 Need
7402 10~15 Need
Hep 3B 10~30 Need
Hep G2 10~30 Need
SMMC-7721 10~30 Need
Huh-7 10~30 Need
Hela 10~30 Need
HOS 20~40 Need
Hep-2 10~30 Need
HL-60 >100 Need
HT-29 10~30 Need
PKO 2~4 Need
SW480 10~30 Need
DLD-1 10~30 Need
SK-OV-3 2~4 Need
SHG-44 10~30 Need
U251 1~3 Need
U87 1~3 Need
293T 1~3 Need
HUVEC-2C 10~30 Need
PC-3 20~40 Need
MDA-MB-231 10~30 Need
MCF-7 20~40 no
Tca8113 10~30 Need
RPE 10~30 Need
AGS 100~150 Need
BGC-823 100~150 Need
SGC-7901 10~30 Need
MKN-28 20~40 Need
MKN-45 20~40 Need
BxPc-3 20~40 Need
CFPAC-1 50~80 Need
Panc-1 2~4 Need
HEC-1-B 2~4 Need
NIH-3T3 20~40 Need
Raw264.7 10~30 no
CHO 20~40 Need
HSC-T6 10~30 no
C6 >100 Need
NRK 10~30 Need

Note: Influenced by cell source, algebra and cell state, the MOI values in different laboratories may be different to some extent. Data in this table are obtained on condition that the cell is in good state, and infection efficiency can reach 85-100% cells.

Day3: Refresh the culture medium 24 hours post infection.

Day4: Change to fresh medium with puromycin 48 hours post infection. The recommended concentration of puromycin ranges from 1 to 10μg/ml according to cell lines. Set the uninfected wild-type cells as control group and add equal volume and concentration of puromycin. Replace with fresh puromycin-containing medium every 2 or 3 days until the control group cells die out. Then choose one of the following steps according to experimental requirements.
1. Non-selecting monoclonal cells Passage the infected cells and select with puromycin constantly. Freeze the cell mixture in continuous three passages. Considering the heterogeneity, we recommend selecting monoclonal cell for a confirmed phenotype.
2. Selecting monoclonal cells Select at least five monoclonal cells after infection and puromycin selection, and propagate in puromycin-containing medium. Detection the expression of target genes using western blot or qPCR. Choose the stable cell line with proper expression level of target genes to passage three generations and freeze the stable cell line.

Notes for infection of special cell lines.
1. Suspension cells
We recommend using flat fillet centrifuging transfection to infect suspension cells or semi-suspension cells. Add virus suspension into cell culture dish, sealing tightly, and centrifuge at low speed of 200×g for 1 hour in the flat fillet centrifuge. Place cells in cell culture incubator after centrifuging transfection. If the flat fillet centrifuge is inaccessible, you can suspend the cells and transfer cells into centrifuge tubes, followed by low-speed centrifuge, and discard the most of supernatant. Add virus suspension into the tubes, resuspending cells, place it at room temperature for 15 min (no more than 30 min), and transfer the cells and virus suspension into plate to culture. Replace with fresh culture medium the next day.
2. Cells difficult to infect
For cells difficult to infect, like DC cells, we recommend repeated infections. Replace with fresh virus suspension 24 hours after the first infection. Repeated infections can increase the infection efficiency markedly.
3. Non-dividing primary cells
We recommend high-titer adenovirus to infect these cells like BMSC.

Detailed information of Lentivirus protocol can be seen in Lentivirus Protocol.