91
Collection (ATTC) guidelines. Cells are commonly stored in liquid nitrogen (−195.8 °C) in special vials and the thawing process
is as follows:
1. Prepare water bath at 37 °C.
2. Submerge the vials for 2–3 min and gently agitate.
3. Disperse them in growth medium.
4. Transfer to cell culture flask.
5. Incubate until it becomes confluent.
The incubation environmental conditions should be at 37 °C
in a humidified atmosphere with 5% CO 2 supply.
The cryo-storage medium is FBS and it is common to be
enriched by DMSO which is used in order to avoid the formation
of ice crystals during freezing that would be detrimental for the
cells. In this case the growth medium where cells were incubated
should be removed as soon as they start to grow in order to avoid
contamination from DMSO.
When cell cultures reach confluency on the first passage the
cells can be detached and used for the biological evaluations discussed below. The detachment protocol involves the following
steps:
1. Remove supernatant growth medium.
2. Gentle rinse with PBS of a volume enough to cover the flask.
3. Remove PBS.
4. Add trypsin of known volume just enough to cover the flask.
5. Incubate for a time depending on the specification of each cell
line to achieve detachment.
Caution! Do not exceed the time limit as trypsin might cause
cell damage.
6. Agitate the flask gently.
7. Add triple volume of growth medium based on the added
trypsin volume and cell suspension.
8. Count alive cells with hematocytometer according to published protocol (see Note 4).
9. Create dilution to reach the desired cellular population for the
applied assay.
For most assays a confluency of 70 or 80% is required after a
24-h incubation.
10. Seed the cell-containing growth medium of known population in the appropriate plate for the experiment.
ΜΤΤ assay is a colorimetric methodology which is based on the
reduction of yellow tetrazolium to violet formazan crystals for
assessing quantitative cell viability when the cells are incubated
with materials, drugs, small molecules, nanoparticles, proteins, and
3.3.2 MTT Cell
Proliferation Assay
Multisensitive Polymeric Nanocontainers as Drug Delivery Systems: Biological Evaluation
Collection (ATTC) guidelines. Cells are commonly stored in liquid nitrogen (−195.8 °C) in special vials and the thawing process
is as follows:
1. Prepare water bath at 37 °C.
2. Submerge the vials for 2–3 min and gently agitate.
3. Disperse them in growth medium.
4. Transfer to cell culture flask.
5. Incubate until it becomes confluent.
The incubation environmental conditions should be at 37 °C
in a humidified atmosphere with 5% CO 2 supply.
The cryo-storage medium is FBS and it is common to be
enriched by DMSO which is used in order to avoid the formation
of ice crystals during freezing that would be detrimental for the
cells. In this case the growth medium where cells were incubated
should be removed as soon as they start to grow in order to avoid
contamination from DMSO.
When cell cultures reach confluency on the first passage the
cells can be detached and used for the biological evaluations discussed below. The detachment protocol involves the following
steps:
1. Remove supernatant growth medium.
2. Gentle rinse with PBS of a volume enough to cover the flask.
3. Remove PBS.
4. Add trypsin of known volume just enough to cover the flask.
5. Incubate for a time depending on the specification of each cell
line to achieve detachment.
Caution! Do not exceed the time limit as trypsin might cause
cell damage.
6. Agitate the flask gently.
7. Add triple volume of growth medium based on the added
trypsin volume and cell suspension.
8. Count alive cells with hematocytometer according to published protocol (see Note 4).
9. Create dilution to reach the desired cellular population for the
applied assay.
For most assays a confluency of 70 or 80% is required after a
24-h incubation.
10. Seed the cell-containing growth medium of known population in the appropriate plate for the experiment.
ΜΤΤ assay is a colorimetric methodology which is based on the
reduction of yellow tetrazolium to violet formazan crystals for
assessing quantitative cell viability when the cells are incubated
with materials, drugs, small molecules, nanoparticles, proteins, and
3.3.2 MTT Cell
Proliferation Assay
Multisensitive Polymeric Nanocontainers as Drug Delivery Systems: Biological Evaluation