Difference between revisions of "Team:Freiburg/Methods"

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<h2>SEAP Assay</h2>
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<p>SEAP (secreted embryonic alkaline phosphatase) assays were performed to measure the induction of the HRE promoter.
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<br>Firstly, 200 µl of cell supernatant was transferred to 1.5 ml Eppendorf Tubes. These were heated for 30 min at 65°C, followed by a centrifugation step (1250 g, 1 min).
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<br>2 µl of 2x SEAP buffer (20 mM homoarginine, 1 mM MgCl2, 21 % (v/v) diethanolamine, pH 9.8) were filled into wells of a 96 well plate. 40 µl of the heated cell supernatant were added to the wells with 2x SEAP buffer.
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<br>The addition of 68 µl of the substrate pNPP (120 mM para-nitrophenyl phosphate dissolved in H2O) was directly followed by spectroscopic measurement for 180 min, every 30 s at a wavelength of 405 nm in a microplate reader (Biotek Synergy H4 Hybrid Reader).
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Revision as of 15:33, 12 October 2017


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Cell Culture

Transfection

Mammalian cell transfection is used to introduce foreign nucleic acids into cells. Depending on cell line and desired effect, there are different ways to transfect mammalian cells. These methods can be broadly classified into two groups: transient and stable transfections. Normally, a selection marker is needed to identify cells that are transfected.

Transient Transfection

Transient transfections generate short-term impact of alterations in gene and protein expression. In this case, the expression of the introduced gene is temporary because the foreign nucleic acids are not integrated into the host cell genome and therefore will not be passed on by cell division. Transient transfection of cells was achieved by two different methods:

PEI transfection

Polyethylenimine (PEI) is used as a polymeric transfection. PEI condenses DNA into positively charged particles, which bind to anionic cell surface residues and are brought into the cell via endocytosis. Using this method transient transfected HEK cells were generated.

Electroporation

Electroporation is a method, which is used to introduce target genes into cells transiently in a physical way. To achieve this, an electrical field is generated around the cells to increase the permeability of the cell membrane, so that DNA can enter the cells. During our project we used electroporation to generate transient transfected Jurkat cells.

Stable transfection

The aim of stable transfection is to introduce a gene of interest into the genome of cells, which results in permanent expression of foreign genes in target cells. Lentiviral transduction is used in this project to generate stable cell lines.

Lentiviral Transduction

Stable transfection via lentiviral transduction delivers the foreign gene into the host’s genome. This ability was utilized in scientific research for constitutive expression of certain gene in the target cells. Lentiviruses are a subtype of retroviruses. Compared to standard retroviruses, lentiviruses are capable of infecting non-dividing and actively dividing cell types while standard retroviruses can only infect replicating cell types. Although viral vectors are derived from pathogenic viruses, their toxicity and the risk of handling them are reduced via modifications. The necessary components for virus production are splitted into multiple plasmids to increase the safety. The 2nd-generation lentiviral system is used in our project, for which one packaging plasmid and one envelope plasmid are needed for the virus production along with the transfer plasmid containing the gene of interest.



Cryo Stocks

To store cells for a long time cryo stocks were made:

HEK cells: HEK cells were washed with PBS and trypsinized. Trypsinization was stopped with RPMI media and the sample was centrifuged
(100 g, 3 min). The supernatant was discarded and the pellet was resuspended in RPMI and the cells counted according to our protocol (LINK?).

After another centrifugation step (100 g, 3 min) the supernatant was discarded and the pellet resuspended in 1 ml of 90% FBS and 10% DMSO.

The cells were transferred to a cryo tube, which was stored for 2 days at -80 °C in an ice box to ensure gradual freezing of the cells. The tubes were then placed into a liquid nitrogen tank for long term storage.

Jurkat cells: Jurkat cells from a full 10 cm dish were transferred to a 15 ml falcon tube and centrifuged (100 g, 3 min). The supernatant was discarded and the pellet resuspended in 2 ml of 90% FBS and 10% DMSO. Aliquots of 1 ml cell suspension were transferred into a cryo tube and stored for 2 days at -80 °C in an ice box to ensure gradual freezing of the cells. Afterwards the tubes were placed into a liquid nitrogen tank for long term storage.

Thawing of the samples:

To thaw samples, they were taken out of the liquid nitrogen tank and placed into a 37 °C incubator. 15 ml falcon tubes with 5 ml RPMI were also placed in the 37 °C incubator to heat up. Afterwards the thawed sample was transferred to the warmed 15 ml falcon tube with RPMI and centrifuged (100 g, 3 min). The supernatant was discarded and the pellet resuspended in RPMI. The sample was then transferred to a 6-well plate and could be placed back into the incubator.

SEAP Assay

SEAP (secreted embryonic alkaline phosphatase) assays were performed to measure the induction of the HRE promoter.
Firstly, 200 µl of cell supernatant was transferred to 1.5 ml Eppendorf Tubes. These were heated for 30 min at 65°C, followed by a centrifugation step (1250 g, 1 min).
2 µl of 2x SEAP buffer (20 mM homoarginine, 1 mM MgCl2, 21 % (v/v) diethanolamine, pH 9.8) were filled into wells of a 96 well plate. 40 µl of the heated cell supernatant were added to the wells with 2x SEAP buffer.
The addition of 68 µl of the substrate pNPP (120 mM para-nitrophenyl phosphate dissolved in H2O) was directly followed by spectroscopic measurement for 180 min, every 30 s at a wavelength of 405 nm in a microplate reader (Biotek Synergy H4 Hybrid Reader).