Calcium Phosphate Transfection

Title: Transfection to microglia and astrocytes via “calcium phosphate” method.

Conducted by: Sagi Angel

Date: 3-7.6.18

Aim: In this experiment we have tried to use the calcium phosphate protocol in order to transfect microglia astrocytes and HEK as control in GFP gene. The use of HEK cells as control, is due to its ability to be transfected relatively easily by the various techniques, including calcium phosphate method.

Importance: This experiment was carried out in parallel with experiments using different methods of transfection (different reagents and electroporation) in order to find an efficient way of inserting our plasmids into astrocytes and microglia for the continuation of the project.

Design:
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Expectations:

The expected results were the illumination of GFP under a fluorescent microscope. It could be assumed that if the experiment was conducted well, the HEK cells would be able to insert the GFP plasmid easily and the illumination would be high, and one could hope that some of the experimental cells, astrocytes and microglia, would also show fluorescence. If there was a problem during the experiment or in the quantities of the materials, it would not be possible to see fluorescence in the HEK cells or we would see a high cell mortality rate.

Results:

1. All of the cell types survived the transfection process –no significant death rate was noticed
2. In the HEK cells used as control indeed there was an expression of the GFP gene under the fluorescence microscope the expression wasn’t high
3. No GFP was detected in the microglia and astrocytes cells in both option of transfection

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Discussion:

The purpose of the experiment was to introduce a fluorescent gene into microglia and astrocytes using the HEK cells as control. Because fluorescence illumination is obtained in the control cells, it is assumed that the transduction process worked properly, but the specific cells of the microglia and astrocytes do not respond well to the process in a manner similar to the control cells. It should be noted that the percentage of infection in the control cells was not particularly high, which may indicate a process that is not done optimally or on problems in concentrations of DNA.

Conclusion:

The process of transfusion using calcium chloride is simple and inexpensive, but does not allow high penetration rates in our target cells - astrocytes and microglia.

While there was fluorescence illumination in the control cells, it was lower than expected, which means that conditions may be improved to increase efficiency, and this may also could have effect on the target cells.

Since there are many materials that make up the buffer, it is possible that a change in quantity or lack of accurate preparation may affect the quality of the results.

It should be concluded that this method, especially in a short period of time that does not allow many adjustments and repetitions, is not suitable for the continuation of work in the project and has indeed been abandoned in favor of the use of ready-made reagents that showed higher efficiency in control cells and astrocytes and microglia.

Transfection by transfection-reagent

Title: Transfection of the C8-D30 astrocytes and BV-2 microglia cell lines with PUC-GFP vector.

Conducted by:Liat Tsoran and Ori Tulchinsky

Date:

Aim: In this experiment we have tried to use jetPEI-Macrophage and jetPRIME transfection reagent in order to transfect microglia cells (BV2) and astrocyte cells (C8-D30), respectively, while using HEK-cell as a positive control. The use of HEK cell-line as a positive control, is due to its transfectability by the various techniques.

Importance: This experiment was carried out in parallel with experiments using different methods of transfection (different reagents, calcium phosphate and electrophoresis) in order to find an efficient way of inserting our plasmids into microglia for the continuation of the project.

Experiment 1 – Microglia (BV2) transfection:

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Experiment 2 – Astrocyte (C8-D30) transfection:

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Expectations:

The expected results were to detect GFP fluorescence under a fluorescent microscope. It could be assumed that if the experiment was conducted well, the HEK cells would be able to harbor the GFP containing plasmid and fluorescence would be high. One could have hoped that some of the cell-lines of our experimetns would also show fluorescence. If there was a problem during the experiment or in the quantities of the materials, it would not be possible to see fluorescence in the HEK cells or we would see a high cell mortality rate.

Results:

1. All the cell types survived the transfection process –no significant death rate was noticed.
2. In the HEK cells used as control indeed there was an expression of the GFP.
3. No GFP was detected in the microglia nor the astrocyte cells in all groups of transfection.

Discussion:

The purpose of the experiment was to introduce a fluorescent gene into microglia and astrocytes using the HEK cells as control. Because fluorescence illumination is obtained in the control cells, it is assumed that the transfection process worked properly, although that microglia and astrocytes did not respond well to the process in a manner similar to the control cells.

Conclusion:

The process of transfection using jetPEI-Macrophage transfection reagent for microglia cells and jetPRIME for astrocyte cells was unsuccessful. For this reason, we switched to other methods - prior to electrophoresis and when this method was not successful we transferred all our vectors to Lantivirus vectors and we used the Lentivirus infection method.

Astrocyte Activation

Title: Activation of astrocyte cell line C8-D30 using lipopolysaccharide (LPS) and pro-inflammatory cytokines.
Conducted by: Mor Sela

Date:  29.7.18-2.8.18

Aim: Activation of astrocytes was performed to confirm that our C8D30 astrocyte cell line can accurately model resting and reactive astrocytes for our experimental design.

Importance:Our project is based on the assumption that reactive astrocytes are a main factor in ALS and therefore our product is designed to specifically disarm them. Without a “reactive astrocyte” experimental group, we can not test the efficiency and specificity of our product in reactive astrocytes when compared to other cells in the system.

Design:

Experiment 1 – Measurement of C3 in activated astrocytes using ELISA

Expectations:

• In negative control 1 the astrocytes were treated with microglia growth medium which did not contain microglia. Then the astrocytes were grown with this medium and with astrocyte growth medium. We expected a negligible C3 concentration in negative control 1.
• In negative control 2 the astrocytes were treated with medium from microglia cell culture (without LPS). Then the astrocytes were grown with this medium and with astrocyte growth medium. We expected a negligible C3 concentration in negative control 2.
• In sample 1 the astrocytes are treated with medium from microglia cell culture with LPS. Then the astrocytes are grown with this medium and astrocyte medium. We expect a high C3 concentration in sample 1.
• In sample 2 the astrocytes are treated with medium from microglia cell culture grown in astrocyte medium with LPS. Then the astrocytes are grown with this medium and more astrocyte medium. We expect a high C3 concentration in sample 2, possibly a different concentration than seen in sample 1 as the microglia are not grown in medium specific to them.

Results:

The calibration curve is close to linearity with an R² value >0.99, meaning that the extrapolated results, such as the unknown protein concentration, are statistically reliable.

The results show a high concentration of C3 in samples 1 and 2. There is no significant difference in C3 concentration between these two samples.

Conversely, a low C3 concentration were found in negative control 1 and 2.  There is a small increase in C3 in sample 2 over sample 1.

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Discussion:

This is the first attempt at activating astrocytes and was performed with LPS added to microglia medium. The purpose of this experiment was initial examination of the reactivity level of C8D30 astrocyte cell line.

The results agree with our expectations. The C3 concentration is lower in the negative controls as opposed to the astrocytes which were activated. These results indicate that the astrocytes were indeed activated and that we can create two experimental groups – resting and reactive astrocytes.

We must note that the negative controls contained a low concentration of C3, possibly because astrocytes secrete a certain amount of C3 when in a resting state. The difference in C3 concentration displayed in the negative controls does not concur with our expectations. We expected there to be no difference LPS (rather than cytokines) is not supposed to activate astrocytes, and therefore the C3 concentrations should be low and similar in both negative controls. However, this can be explained by the lack of biological repetitions. As only one biological repetition was performed for the negative controls, we can not be sure there was indeed a significant difference in concentration between them.

Experiment Weaknesses:

• We are missing a negative control of astrocytes grown in their medium with no treatment. Without this sample we can not make a reliable comparison of C3 between activated and resting astrocytes.
• Not enough and unequal biological repetitions for all samples.
• Missing additional comparative parameters such as astrocytes activated using commercial cytokines, incubation for different periods of time, etc…
• The ELISA kit is very expensive; therefore, we were limited in the repetitions we could perform for this experiment.

Experiment Strengths:

• ELISA is a very precise, sensitive method which produces quantitative results increasing the reliability of these results.