Molecular Cell Biology And Physiology Assessment Sample

Introduction of Molecular Cell Biology And Physiology Assessment

This study has been comprised of 3 major experiments in the field of molecular biology in terms of determining and evaluating the impact of H₂O₂ with respect to the viability of cells. Thus, the cell lines of SHSY5Y neurons have been chosen for the determination of viability in contrast to H₂O₂. The experiment has been performed based on the uptake of peroxidase (HRP) by the neuronal SHSY5Y. Thus, it can be stated that the transformation of cell response due to the uptake of HRP can be measured. This type of experiment has been performed through the "Trypan Blue Exclusion Assay" method thus visibility of H₂O₂ over the neural cell lines can be noticed (Ferlazzo et al. 2020). On the other hand, the toxicity level of H₂O₂ on SHSY5Y neurons can be noticed through the XXT assay method. Therefore, the objectives of this study have been contemplated below.

• To explore the impact of H₂O₂ on SHSY5Y neuronal cell viability determination through the "Trypan Blue dye exclusion" method.
• To observe the uptake of "Horseradish Peroxidase" by the cell lines of SHSY5Y neuronal determination through extraction and measurement of peroxidase activity. 
• To understand the impact of H₂O₂ on SHSY5Y neuronal cell viability pretreated with peroxidase and determination through XXT assay. 

SHSY5Y has been recognised as a "Thrice-subcloned" type of cell line generated from the cell lines of "SK-N-SH neuroblastoma". This has a broad-scale use in terms of practising experimental studies of neurology with respect to physiological and pathological features (Laurutis et al. 2022).  In terms of performing such experiments, seeding of SHSY5Y cells need to be performed. However, seeding density refers to a significant variable in the case of the functional approach of tissue engineering thus 24-well and 6-well plates have been considered (Ham et al. 2020). Their seeding density is respectively 5 x 10⁵ and 2 x 10⁶ cells/well. Those cultures need to be incubated for a period overnight in terms of specific and efficient compilation of the activity of H₂O₂ against SHSY5Y. 

It has been observed that H₂O₂ has been considered an oxidizing agent and have the major capability in term of inducing damage to cells. Thus, the use of inappropriate concentrations over the cell lines might leads to the arrest of cell progression with respect to the cell cycle. Along with that higher concentrations may result in the apoptosis of cells through oxidizing a group of thiol proteins. The mitochondrial pathway of the cell has been triggered by H₂O₂ and results in loss of caspase-3 activation. Therefore, cell lines needed to be infused with H₂O₂ and incubated thus a large number of cells on different concentration levels will be dead. The visibility of dead cell lines needs to be performed using the stain or dye. Thus, it can be stated that dead cell lines of SHSY5Y can be stained with Trypan Blue and the impact of H₂O₂ at different concentrations with respect to SHSY5Y neuronal cells can be noticed.   

Besides that, the uptake of HRP by the cell lines of SHSY5Y takes place through retrograde transport by axons and it has major significance in the field of pathology and physiology of neurons. Kristensson & Olsson in the year 1976 coined out that chromatolytic activity of the neurons has been induced by "Retrograde transport" with respect to the region of injury. Thus, HRP has major acceptance as a tracer substance in the segment of "Retrograde axonal transport" (Barra et al. 2022). Therefore, it can be stated that measurement of peroxidase activity can guide through the exploration of the uptake of "Horseradish Peroxidase" with respect to the neural cell lines "SHSY5Y". 

It has been observed that the exploration of dopaminergic pathogenesis has been performed with SHSY5Y cells. The cell lines of SHSY5Y have the capability to express representative markers of dopaminergic that includes tyrosine hydroxylase. Thus, it can be stated that the effect of H₂O₂ has the potential to influence tyrosine hydroxylase. Therefore, cell lines have been recognised as a suitable model to study the effect of H₂O₂ in terms of determination by counting dead cells with respect to different concentrations (Pap et al. 2022). Hence, XTT assay has been considered in terms of measurement of the activity of "Dehydrogenase Enzyme".

This assay is based on the capability of viable cells in order to convert "5-Diphenyltetrazolium Bromide" and "3-[4, 5-Dimethylthiazol- 2-yl]-2" into an orange-coloured formazan dye (Kruni? et al. 2021). The concerned assay has been performed with respect to the viability of mitochondria thus survival of cell lines of SHSY5Y can be noticed against to H₂O₂ compound.

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Result of experiment on cell lines of SHSY5Y neurons 

Experiment 1

In this experiment 1 of the impact of H₂O₂ is identified by Trypan Blue dye exclusion that is helped to determine the SH-SY5Y neural cells. It is estimated that the plates of this experiment have been seeding the whole night at the minimum temperature of 37ºC (Hoet al. 2022). In this experiment, it is noticed that there are three types of plates used in different sizes such as 0.1, 0.2, and also 1.0 mM for approximately one whole day with H₂O₂. On the other hand, in experiment 1, there are five types of cells that are used to get the best result 3 nM repeat, 3 uM repeat, 30 nM repeat, 30 uM repeat, and 300 nM repeat. Therefore, all the result of those cells has been tested with repeat 1, 2 and 3. [Referred to Appendix 1]

Thus, the viability of the cells is examined by using the MTT assay and also for delivering the control cells which are untreated. Therefore it is found that the ANOVA is conveyed for the breakdown in the experimental segment of statistical ground (Sirinupong et al. 2021). There are three types of results are come from the experiment repeat 1, repeat 2 and repeat 3. Hence, the result from the 3 nM Repeat 1, repeat 2 and Repeat 3 has been analyzed. On the other hand, the H₂O₂ impact in these cells by excessive exposition of oxygen metabolites including H₂O₂ leads to the disease as well as oxidative stress.  [Referred to Appendix 2] 

On the other hand, the neutral cells are approximately provided with the result that is shown in the images. In the case of the concentration of H₂O₂ being high then the activities of cells are also decreasing. The number of dead cells is increasing as a result. Therefore, it is estimated that in some experiments there are a wide number of black spots are shows that refer to dead cells. The black spots are increased because of the high concentration of H₂O₂. Therefore, it is shown that in the 30 uM repeats 1, 2, and 3 have the most black spots than the other cell cultures.  [Referred to Appendix 3] Thus, this experiment has shown that in the 3 uM repeat cells there is the lowest number of black spots. 

Experiment 2

In experiment 2, there are three types of plates are used for getting the best result which is the uptake of Horseradish Peroxidase by SHSY5Y neuronal cells specified by extraction and also dimensions of peroxidase activity (Zacharia et al. 2022). Control cells and HRP-treated cells are the main two types of this experiment. The graph of this plate 1 is shown in the graph that refers to the almost same bars. Therefore, the HRP-treated cells of plate 1 are calculated with the highest bar in sample 2 than the other cells in this experiment.  [Referred to Appendix 4] On the other hand, in plate 2, the graph bars of the control cells are found almost in the same position near about 0.3 on the Y axis. 

Cell Samples
Control cells	0.254	0.299	0.273	0.257	0.284	0.258	0.252	0.247	0.239	0.255
HRP treated cells	0.656	0.678	0.623	0.652	0.671	0.653	0.649	0.651	0.651	0.697

Table 1: Plate 1 of experiment 2

(Source: Self-developed)

In plate 2, there are also two types of cells as in plate 1. Thus, the control cell on the 2 number has the maximum height of the bar than the other bars.  [Referred to Appendix 6] On the other hand, in the number 10 of the X axis, the HRP-treated cells have the maximum height of the bar. 

Cell Samples
Control cells	0.246	0.293	0.266	0.249	0.277	0.250	0.244	0.238	0.230	0.247
HRP treated cells	0.668	0.691	0.633	0.664	0.684	0.665	0.660	0.663	0.663	0.711

 Table  2: Plate 2 of experiment 2

(Source: Self-developed)

The HRP-treated cells displayed the outcome with the most elevated bar in cell models 2, 6, and 10. In experiment 3, the graph of the control cells shows the highest bar in samples 1, 5 and also 9.  [Referred to Appendix 5] Thus, in the bar of the graph of HRP-treated cells has the precise picture that shows the highest image. 

Cell Samples
Control cells	0.252	0.295	0.270	0.255	0.281	0.256	0.250	0.245	0.237	0.253
HRP treated cells	0.638	0.659	0.606	0.634	0.652	0.635	0.631	0.633	0.633	0.677

 Table 3: Plate 3 of experiment 2

(Source: Self-developed)

Experiment 3

In experiment 3, there are three plates are used for H2O2 on the viability of SHSY5Y neuronal cells pretreated with HRP determined by XTT. The XTT cells are calculated and on the plates, the cell samples are incubated the whole night at 37ºC temperature.  [Referred to Appendix 7] Hence, the absorbance data from the experiment of XTT assay is provided in the analysis of myBeckett. Therefore, there are three plates that show three different types of outcomes that are occurring between Control cells and HRP-treated cells. 

H2O2conc (µM)	0	3.9	7.8	15.6	31.3	62.5	125	250	500	1000
HRP treated Cells 1	1.962	1.537	1.375	1.009	0.874	0.783	0.701	0.624	0.354	0.275
HRP treated Cells 2	1.984	1.605	1.437	1.102	0.821	0.807	0.652	0.609	0.375	0.301
HRP treated Cells 3	1.903	1.781	1.298	1.097	0.862	0.755	0.729	0.567	0.341	0.319
Control Cells 1	2.931	1.263	1.009	0.775	0.637	0.392	0.307	0.301	0.284	0.289
Control Cells 2	2.978	1.284	1.107	0.801	0.623	0.451	0.315	0.263	0.312	0.273
Control Cells 3	2.952	1.107	1.056	0.834	0.576	0.482	0.278	0.319	0.303	0.327

Table 4: Plate 1 of experiment 3

(Source: Self-developed)

In plate 1, there are three types of results that come from Cells 1, cells 2, and cells 3. In plate 1 of experiment 3, the HRP-treated cells 1 and 3 Vs the Control Cells 1 and 3 illustrate the fast deduction of the sequence 1 and 2 in the graph.  [Referred to Appendix 8] Therefore, cell 2 of both control cells and HRP treated deliver some up points of series 2 in the 3.2 ends of the graph. 

H2O2conc (µM)	0	3.9	7.8	15.6	31.3	62.5	125	250	500	1000
HRP treated Cells 1	1.807	1.437	1.296	0.978	0.860	0.781	0.710	0.643	0.408	0.339
HRP treated Cells 2	1.826	1.496	1.350	1.059	0.814	0.802	0.667	0.630	0.426	0.362
HRP treated Cells 3	1.756	1.649	1.229	1.054	0.850	0.757	0.734	0.593	0.397	0.378
Control Cells 1	2.650	1.199	0.978	0.774	0.654	0.441	0.367	0.362	0.347	0.351
Control Cells 2	2.691	1.217	1.063	0.797	0.642	0.492	0.374	0.329	0.371	0.338
Control Cells 3	2.668	1.063	1.019	0.826	0.601	0.519	0.342	0.378	0.364	0.384

Table 5: Plate 2 of experiment 3

(Source: Self-developed)

In plate 2, the outcome of the selected cells delivers nearly the same in the bar diagram of the result. It is estimated that series 1 of the cells 3 graphs has been shown in the X axis. Control cells and HRP-treated provide a quick deduction in the graph.  [Referred to Appendix 9] Corresponding to this, cell 2 also gives the same result with immediate deduction in its graph. On the other hand, cell 3 delivers the tallest peak of series 3 in the 4.5 line of the graph. Thus, this experiment of this H2O2 on the viability of SHSY5Y neuronal cells delivers the absorbance of plates at 37ºC. 

H2O2conc (µM)	0	3.9	7.8	15.6	31.3	62.5	125	250	500	1000
HRP treated Cells 1	1.987	1.528	1.353	0.958	0.812	0.714	0.625	0.542	0.250	0.165
HRP treated Cells 2	2.011	1.601	1.420	1.058	0.755	0.740	0.572	0.526	0.273	0.193
HRP treated Cells 3	1.923	1.791	1.270	1.053	0.799	0.683	0.655	0.480	0.236	0.213
Control Cells 1	2.705	1.404	1.130	0.877	0.728	0.463	0.372	0.365	0.347	0.352
Control Cells 2	2.956	1.427	1.236	0.905	0.713	0.527	0.380	0.324	0.377	0.335
Control Cells 3	2.628	1.236	1.180	0.941	0.662	0.561	0.340	0.385	0.367	0.393

Table 6: Plate 3 of Experiment 3

(Source: Self-developed)

In plate 3, there are also three types of graphs that deliver three types of cell samples of control cells and also HRP-treated cells.  [Referred to Appendix 10]Cell number 2 has the highest bar in the 3 number of the X axis than the other two graphs of this experiment. 

Discussion

Experiment 1 

It has been observed that SHSY5Y was accurately generated from SK-N-SH cell lines and used through undifferentiated conditions thus both adherent and floating cells were present. Thus, this experiment has been focused to explore the impact of H₂O₂ over the concenred cell line. In this experiment, cell samples were seeded over the 5 x 10⁵ cells/well and this type of seeding has a major benefit with respect to the functional approach of tissue engineering (Zacharia et al. 2022). The cell culture plate was left for incubation for a minimum duration of overnight thus activity of the concerned element over the cell line can be accurately performed.  [Referred to Appendix 4]

It has been observed that different concentration gradients of H₂O₂ have different categories or levels of impact thus a series of concentrations have been considered. Thus, it can be stated that 3nm, 30nm and 300nm along with 3µM, 30µM and 300µM have been considered. Hence, different outcomes can be noticed with respect to the concerned cell lines and the most effective concentration gradient can be considered. Besides that, H₂O₂ have been diluted with respect to a serum-free medium for the development of different concentration gradient (Kaokaen et al. 2022). The major reason behind this scenario reflects avoiding the local formation of high-level concentrations of hydrogen peroxide across the external site.  [Referred to Appendix 5]That might lead to confounding effects thus outcome of this experiment will become much more vast and reproducible. 

Experiment 2

It has been observed that HRP have a major role in this experiment in terms of the conversion of phenolic compounds. This experiment has been performed with the help of an extraction buffer thus a major benefit with protein stability has been gained. The extraction or assay buffer has played a critical role in terms of enhancing the stability of the protein relied on in the concerned experiment. Therefore, the isolation of a specific protein from the region of non-soluble cells of SHSY5Y can be derived (Tiwari et al. 2022). Thus, the development of a standard solution with HRP was essential in terms of gaining effective and accurate outcomes for this study.  [Referred to Appendix 5]

The experiment has featured two distinct types of solutions such as substrate solution and stop solution. It has been observed that substrate solution has a major role in terms of estimating the enzymatic reaction rate of the concerned experiment. Along with that binding affinity of HRP also can be measured with this type of approach. Besides that, "Stop Solution" also has a major significant role with respect to this experiment. It has been observed that the concerned solution has a critical role in terms of suspending the reaction relying on enzyme substrate. The activity of stop solution has been brought into practice after retaining susceptible colour intensity over the sample containing culture wells.  

The concerned cell samples have been exposed to the solution of Phosphate buffer with a gradient value of 100nm. The core reason behind this scenario was to break down the membrane of cells of SHSY5Y through alteration of pH. Thereafter, cell samples have undergone the procedure of vortexing thus sample can oscillate rapidly (Ganguly et al. 2022). After that, samples have been centrifuged thus distinguish of molecules comprised within the sample culture can be accurately performed and the OD of each sample was taken. Thus, a standard curve with respect to HRP depending on cellular samples of SHSY5Y has been obtained.  [Referred to Appendix 9]

Experiment 3

This experiment has been beginning with the seeding of the SHSY5Y cell sample over the "96-well plates" having the technical configuration with "1 x 105 cells/well". The well plate containing the concerned cells has been incubated with HRP for a minimum period of overnight. However, it can be stated that the temperature of the incubation was nearly 37ºC thus no such generic change over the chemical properties of the cellular constituents can be noticed rather than susceptible or desired changes. Thereafter, well plates have been washed with the "Serum-free medium" thus the undesirable constituents can be wiped or removed from the concenred cell sample culture (Liu et al. 2022).  [Referred to Appendix 11] In terms of observing the effect of H₂O₂ over the concerned cell sample, nearly a 1 mM concentration gradient of H₂O₂ has been added over the sample. 

The cells have been treated with a different concentration gradient of H₂O₂ in terms of measuring broad-scale and low-scale intensity of the H₂O₂. It has been observed that well plates containing the cells have been again incubated with XXT for a period of overnight. However, it can be stated that XXT has a major essential role in terms of measuring or estimating of metabolic activity of the cells (Vulin et al. 2022). Thus, XXT has been added to the SHSY5Y cell sample in terms of measuring the cellular activity against the engagement of H₂O₂. Hence, the viability of the cells has been indicated with concurrent metabolic activity with respect to XTT assay (Aung et al. 2022). After that, the optical density of each well-containing sample has been measured in terms of the effective compilation of the concerned experiment.

Conclusion 

It can be concluded that three experiments with respect to the impact of H₂O₂ over "SHSY5Y neuronal cells" viability have been accurately carried out. It has been observed that the medium was alone in the control plate in terms of contrasting the efficacy of chosen oxidation processes of H₂O₂. The stained region of the graph with Trypan blue has projected the dead cells of SHSY5Y. Thus, it can be stated that those concentration levels of H₂O₂ have gone through oxidation activity across the mitochondria of cells.

The solubility nature of HRP has major capability in terms of the production of the signal thus a marker molecule can be labelled with an antibody of secondary nature. The outcome has reflected that a high level of intensity of H₂O₂ over the SHSY5Y has been noticed with the activity of uptake of "Horseradish Peroxidase". The OD at 450nm wavelength has been considered for that sample due to the visibility prospect to the animal eukaryotic cell. Hence, it can be stated that a positive impact of H₂O₂ with respect to "SHSY5Y neuronal cells" viability has been noticed with pretreated HRP by using XXT. 

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