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.

Experiment 2 – Measurement of C3 in activated astrocytes using Western Blot

Design:

 Expectations:

• In sample 1 the astrocytes are treated with microglia medium which did not contain microglia or LPS. We expect a very faint band in the gel was these should be resting astrocytes which secrete a small amount of C3.
• In samples 2 and 3 the astrocytes are treated with medium from microglia cell culture with LPS for 24 and 48 hours, respectively. We expect very bold, thick bands with sample 3 slightly stronger than sample 2.
• In samples 4 and 5 the astrocytes are treated with medium from microglia cell culture grown in astrocyte medium with LPS for 24 and 48 hours, respectively. We expect very bold, thick bands as well. The band may be weaker than samples 2 and 3, since the microglia are not grown in their native medium and therefore may not survive as well leading to lower cytokine secretion. Alternately, the band may be stronger than samples 2 and 3 and the astrocytes grow in only their native medium and therefore will survive better and secrete more C3.
• In sample 6 the astrocytes are grown in their native medium with out any treatment. Therefore, we expect a vary faint band which will correspond to the amount of C3 secreted by resting astrocytes.
• In samples 7 and 8 the astrocytes are treated directly with TNFa, IL1a and C1q cytokines for 24 and 48 hours, respectively. We expect very bold, thick bands in these samples.

 Results:

The bands for all of the samples appear at 120kD, which is the molecular weight of the alpha subunit of the C3 protein (the long chain)¹. In some of the lanes (7-15) ,there also appear faint bands at the 45kD and 13kD height. These sizes correspond to fragments of C3 which are produced after digestion by β-mercaptoethanol (which is used during western blot analysis – adding a "Sample Buffer") .

The bands in lanes 2,3,4,5,6,7,8,14,15 present very similar color and width.

The two biological repetitions of sample 5 (lanes 9 and 10) do not match, rather they appear to have different color and width.

Lanes 11 (sample 6), 12, and 13 (sample 7) appear bolder and thicker than the rest of the bands in the gel.

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Discussion:

The purpose of this experiment was to examine the level of reactivity in C8D30 astrocyte cell line. Some of the results obtained did not comply with our expectations. We expected bands 2 and 11, which correspond to the negative controls (samples 1 and 6), to appear significantly less bold than the other bands. Instead, the negative control results appear like the experimental samples. Furthermore, band 11 (astrocytes which did not receive any treatment) appears very similar to bands 12 and 13 (astrocytes activated by cytokines).

Bands 12 and 13 (astrocytes activated by cytokines) do appear as expected, as the bands are very thick and bold when compared to the negative control in band 2 (sample 1).

The difference in the band strength in lanes 2 and 11 (11 is stronger) indicates that it is preferable to grow astrocytes exclusively in their native medium, rather than in medium intended for microglia.

The different activation methods do not significantly increase the reactivity of the astrocytes. Possibly, adding the three cytokines increases the secretion of C3 protein in the astrocytes and therefore a stronger band appears in this sample (bands 12 and 13). However, the difference in band width is not significant enough to make concrete conclusions.

We repeated this experiment several times (5) with similar analysis (western blot) with similar results, therefore we conclude that these results are reliable and not results of technical or human error.

From these results we must conclude that cell line C8D30 is in a state of reactiveness, without additional induction of activation. Although this does not match the expectations which we based on Liddelow, 2017 these results are not so surprising, as the article is based on work with primary cells while we are working with cell lines.

In conclusion, we can proceed with experiments on our C8D30 astrocyte cell line as a reactive astrocyte model without performing activation treatments. The problem with these results is that we do not have a resting astrocyte model to work with.

 Experiment Weaknesses:

• We used astrocyte cell line rather than primary cells which were used in article from which we took our protocol.
• LPS was added to microglia cell line to induce cytokine secretion, rather than LPS injection to mouse models as in the article.
• Western blot analysis is less precise and sensitive than ELISA. Additionally, the results are qualitative rather than quantitative.

Experiment Strengths:

• The experiment we discussed is representative of four other similar experiments. Therefore, we base our conclusions on many repetitions which indicate that the C8D30 cell line is in a reactive state.

Future Plans:

• GFAP is also considered a marker of A1 reactive astrocytes³. Therefore, we recommend using both C3 and GFAP as markers to strengthen the reliability of experimental results.
• We recommend using ELISA rather than western blot analysis to measure reactivity.
• It is recommended to perform immunohistochemistry with markers for reactivity in the specific cell line used.

 Experiment 3 – Identification of reactive astrocytes using immuno-staining:

The previous methods showed opposing results. The ELISA results indicated that LPS activates C8D30 astrocytes, while the western blot analysis results indicated that LPS does not significantly increase the cell line reactivity. Therefore, we decided to perform a third experiment to confirm whether or not the C8D30 cell line is in a reactive state.

We organized a collaboration with Dr. Dinorah Friedmann-Morvinski from Tel-Aviv university. The DFM lab specializes in combines cell biology, molecular biology, biochemistry, immunology, cancer research, as well as advanced genome-wide techniques and microscopy. The DFM lab performs immune-staining to confirm reactive states in glioblastoma and therefore were well equipped to assist us with this matter, as our lab does not possess the necessary equipment. The DFM lab performed immune-staining on a sample of our C8D30 astrocyte cell line.

Results:

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GFAP and Nestin, both reactivity markers, were found and appear in the same location as the cells stained with DAPI.

Discussion:

The immune-staining results confirm the results we obtained in through western blog analysis. The C8D30 cell line is always in a reactive state. Apparently, the differences in C3 concentration we found when using ELISA were negligible and would have been rejected with enough repetitions.

Conclusion:

The C8D30 astrocyte cell line is in a reactive state without any additional treatment. In order to support this conclusion, we recommend using ELISA with many biological repetitions and biomarkers.

References:

1. Third Component of Complement (C3): Structural Properties in Relation to Functions (topology of functional sites/physiologic C3 fragments/C3-membrane interaction/immune adherence/enzyme subunit) VIKTOR A. BOKISCH, MANFRED P. DIERICH, AND HANS J. MULLER-EBERHARD Department of Molecular Immunology, Scripps Clinic and Research Foundation, La Jolla, California 92037 Contributed by Hans J. Muller-Eberhard, March 10, 1975.
2. Liddelow, S.A., Guttenplan, K.A., Clarke, L.E., Bennett, F.C., Bohlen, C.J., Schirmer, L., Bennett, M.L., M€unch, A.E., Chung, W.S., Peterson, T.C., et al.(2017). Neurotoxic reactive astrocytes are induced by activated microglia. Nature541, 481–487.

3. "Reactive Astrocytes: Production, Function, and Therapeutic Potential"Shane A. Liddelow1,* and Ben A. Barres1,Department of Neurobiology, Stanford University School of Medicine, Stanford, CA 94305, USA,Correspondence: liddelow@stanford.edu (S.A.L.), barres@stanford.edu (B.A.B.),J Neurosci Methods.;1995 May;58(1-2):181-92.