Well. When I quantify lentivirus expression, I use FCS ;-) Anyway, just shove it on the 780 and do a 5x5 tile scan, this will give you a good estimate of MOI.

What can't be done by FCS nowadays? :) I guess you are looking at live cells right (I have noticed fixing decreases the signal)? Many thanks! 

I am afraid that you must have that 514 nm Laser in your instrument. 488 nm does not do any good, but if you can manage with some approximation then it might be alright to use.

Hello 

Many factors can affect transduction efficiency. Not all viral particles floating in

culture medium can eventually transduce (or infect) the cells. Some additives

such as polybrene can enhance the transduction efficiency. But cell type is the

main factor to determine the transduction efficiency. An actively dividing cell line

gives much higher transduction rate than non-dividing cell types. If you

transduced non-dividing cells, a higher Multiplicity of Infection (MOI) has to be

used for your optimal expression.

MOI is the average copy number of lentiviral particles per genome of target cell in

the infected cell population. Since not all particles can be infected cells, the MOI

does not directly correspond to percentage of infected cells. MOI is calculated by

counting the number of the cells and the number of the viral particles to be

transduced. The number of viral particles per cell is defined as multiplicity of

infection (MOI). A higher MOI generates more integration and as a result, higher

level of expression. To obtain optimal expression for your specific application, a

range of MOIs (e.g. from 1 to 20) should be tested. For example, to achieve

single copy integration, theoretically the MOI has to be used at less than one

(such as e.g. MOI=0.3). Practically, at MOI =0.3, dependent upon the cell types,

only 5% ~ 20% cells will be transduced and the majority of transduced cells

should only have one copy of insert. For most cases, you may want to simply add

50ul of our premade LVP into one well of a 24-well plate without worrying too

much about MOI.

1- protocol for Adhesive cells:

Day 1: Remove the culture medium. Add fresh, warm, complete medium

(0.5 ml). Thaw the pre-made lentiviral stock at room temperature. Add the

appropriate amount of virus stock to obtain the desired MOI. Return cells to

37°C / CO2 incubator. (Try to avoid thaw and freeze cycles for pre-made

lentivirus. If you cannot use all virus at one time, you still can re-freeze the

virus at -80°C for future use. But virus titer will decrease by ~10% for each rethaw.)

Day 3: At ~72 hr after transduction, check the transduction rate under a

fluorescent microscope with a suitable filter, or calculate the exact

transduction rate via Flow Cytometry System (FACS) or any other flow

cytometry system (such as Guava machine).

Day 3 + (optional): Transduced cells can be sorted out via FACS, selected by

its specific antibiotics. A pilot experiment should be done to determine the

antibiotic’s kill curve for your specific cell line. (Refer to any literature about

how to generate stable cell lines.)

Day 3+ (optional): Functional assay for your target in transduced cells.

A quick application protocol is: add 50 µl virus into one well of a

24-well-plate where cell density is at 50% ~ 75%. At 72 hours after

adding the virus (no need to change medium during the course),

visualize the positive rate under a fluorescent microscope. For

stable cell line generation, transfer the cells into selective antibiotic

containing medium, or sort the cells through their fluorescent signal

and then select the cells by antibiotics.

Note 2: For some cell types such as primary cells it may take a longer time

for maximal target expression; in some cases, maximal expression

may not be detected until 1 week post-transduction.

2. protocol for Suspension cells:

a- Grow your cell in your complete suspension culture medium, including

shaking if required in a CO2 incubator

b- Measure cell density. When cells reach about 3 x 106

cells/ml, measure

cell viability which should be > 90%, then dilute cells to 1 x 106

cell/ml in

complete medium

c- Transduction: thaw lentiviral particles at room temperature. Simply add

premade lentiviral particle into the diluted cells at a ratio of: 50µl or 100µl

virus per 0.5 ml cells (Note: depending on the cell types; you may need to

use more or less viruses). Grow cells in a flask in the CO2 incubator with

shaking if necessary.

d- At 24 hours after transduction, add an equal amount of fresh medium

containing relevant antibiotics (Note: each type of particles contains an

antibiotic marker and the amount of antibiotic to be used depends on the

cell types). Grow in CO2 incubator.

e- At 72 hours after transduction, check fluorescence under the microscope

or calculate the transduction efficiency using cell sorting machine (like

FACS or Guava machine).

f- You can sort the fluorescent positive cells, and maintain the antibiotic

selection to generate stable cell lines.

i hope help you , Good Luck