1. Introduction
1.1 Overview of the experiment
Cell culture is a process by which cells were grown in comfortable conditions, mainly outside the natural environment. Cell signaling is a process that can be used for cellular communication within the body by releasing as well as receiving hormones by the cells.
As per the view of Reshetnyak et al. (2018), GPCR or G protein-coupled receptor drugs are the biggest as well as the most diverse group of membrane receptors present in eukaryotes. LTK cell line is known, as LTK (Leukocyte Tyrosine Kinase) is a noticeable receptor that belongs on the insulin receptor family. Here in this assignment, it has been focused to give a brief description of the process of cell culture as well as the effect on GPCR drugs on LTK cell lines.
1.2 process of cell culture
Cell culture is a process, which involves several processes in it that can be done in several laboratories for doing several experiments that help in future. As per the view of Schiedel et al. (2018), the cell culture was done for experimenting with several examinations that help to give appropriate data to detect accurate results. Processes of cell culture as follows-
Harvest cells: First, the required cells should be identified for the experiment as well as the characteristics should be matched to the required cell characteristics.
Isolate cells: As mentioned by Kim, D. and Johnson, (2018), the required cell should be isolated from animals or plants as well as preparing it for use in the culture process.
Appropriate growth media: The growth media of the culture process should include several ingredients such as temperature, carbon, glucose, amino acids, vitamins and salt solution for growing these cells properly.
Cell incubator: Then, the cells should place into an incubator as well as given proper temperatures for growth of these cells in a proper way (Insel et al. 2012).
Verify: As per the view of Hasenoehrl et al. (2018), after that identified the cells which are used in further examination in this cell culture process.
Figure 1: Cell culture process
(Source: Hasenoehrl et al. 2018)
1.3 LTK-cell line
LTK cell line or Leukocyte tyrosine kinase is a receptor, which belongs to the superfamily of insulin receptors as well as mainly expressed on the pre-B lymphocyte and the neuronal tissue.
As per the view of Lobingier and von Zastrow, (2019), identify the reaction on the LTK cells; these cells are retained from the endoplasmic reticulum of the animal. A membrane protein can be presented on an integral part of the membrane, endoplasmic reticulum, as well as on the plasma membrane. These LTK cell lines are sensitive due to the reaction of GPCR drugs and showing a result of a 1.4 Functions of GPCR drugs
GPCR drugs are the largest as well as the most diverse group of membrane receptors, which release an active form of G-proteins surface on the receptor.
GPCR drugs include both antagonists and agonists, which are used in several disease treatments of major organ systems, including the cardiovascular, central nervous system (CNS), respiratory, urogenital systems. As influenced by Mo et al. (2017), it has several functions as follows-
- It mediates individuals’ senses, such as smell, taste, and vision that help to provide several health benefits to them.
- These drugs are also involved in cell recognition as well as a communication process that helps to emerge from their activity as a superfamily of drugs (Okeke et al. 2019).
1.5 Mechanism of GPCR drugs on fibroblast cell
Cancer-associated fibroblast cells are abundantly present in the body as well as contribute the extensive fibrotic stroma in the body. As influenced by Schiedel et al. (2018), G-protein coupled receptors also affect these fibroblast cells as well as regulate their pro-fibrotic phenotype as well as communicate between cancer-associated fibroblast and pancreatic ductal adenocarcinoma. It also helps to assess the GPCR expression on the normal pancreatic stellate cell converted to cancer-associated fibroblast cells.
1.6 Aim of the experiment
This experiment aims to give a brief description of cell culture that helps to give a clear picture of its process as well as helps in future research. This assignment also gives a brief description of GPCR drugs and LTK cells as well as the impact of GPCR on the fibroblast cell in a lab. The aim of this study also helps to give clear pictures of this assignment that helps to promote the experiments in future as well.
2. Result
2.1 Description of the aim
The aim of this assignment gives a brief description of cell viability calculation that helps to identify the count of cell viability during cell culture. Also, give accurate data on the cell number calculation that helps to precede this experiment in a proper way.
Table 2 gives confluence data, which helps to determine the effect on these two GPCR drugs such as HP7 AND XB7 on the LTK cell lines. The graph of confluency data that helps to recognized the process of this experiment appropriately.
2.2 Table 1
| Drugs name | Concentration (mM) | Corner square | Viable Cells | Dead Cells | Total cells | |
| HC7 | XB7 | 35 | 1 | 44 | 8 | 52 |
| 2 | 46 | 4 | 50 | |||
| 3 | 39 | 2 | 41 | |||
| 4 | 26 | 8 | 34 | |||
| Mean values | 38.75 | 5.5 | 44.25 | |||
Table 1: Cell count data
(Source: Created by learner)
2.3 Cell number and viability calculation
The mean cell number= Cells/ml= N x D x (1/V)
=38.8 x 2 x 104 =7.76 x 105 cell/ml
N is mean number of number of viable cells/corner square
V is liquid volume over the corner square inside cm3 (V=10-4 and 1/V=104)
Dis factor of dilution for trypan blue
Cell viability (%) = mean live cell x 100 = 38.8 x 100 =87.6%
Mean total cell (alive + dead) 44.3
Volume required (ml) = cell number per well = 1 x 105 = 0.12 ml
Cell/ml in cell suspension 7.76 x 105
Volume medium required (ml) = 1.8-0.12 =1.68 ml
Final concentration calculation:
C1V1 =C2V2
35 mM x 0.2 ml = C2 x 2 ml
C2 = 35 mM x 0.2 ml =3.5 mM
2 ml
2.4 Table 2
| Confluency after crystal violet staining (%) | Overall appearance of stained plate | |
| Control (a) | 50 | Purple with pale patches |
| Control (b) | 45 | Purple with pale patches |
| Drug 1 (a) | 75 | Purple with some pale patches |
| Drug 1 (b) | 85 | Mostly purple stain |
| Drug 2 (a) | 45 | Purple with pale patches |
| Drug 2 (b) | 40 | Purple with pale patches |
Table 2: Confluency data
(Source: Created by learner)
2.5 Graph of Confluency data
2.6 Description of figures and tables
Table 1 has been given an n accurate number of viable cells as well as dead cells, and it can be observed that viable cells are increased in number than the dead cells. The calculation of Table 1 gives the final concentration amount that is 3.5mn, which affects cell viability and cell death. Table 2 the Confluency data gives the data on confluency percentage after staining with crystal violet as well as two GPCR final drug concentrations on the staining of these LTK cell lines.
3.1 Summary of the experiment result
This experiment helps to give proper knowledge about cell viability and cell death in a cell culture process. It also helps to give a confluence data depending on the activity of two GPCR drugs on the LTK cells that help to support the experiment data that help to support it for further research in future.
3.2 GPCR signaling mechanism
G-protein-coupled receptors are the biggest as well as the most noticeable group of receptors on a membrane in eukaryotes that mainly persist in the plants. These G proteins are the special proteins with an ability to bound nucleotides GDP (guanosine diphosphate) and GTP (guanosine triphosphate). As per the view of Kim and Johnson, (2018), G- a protein in it which is a heteromeric structure that contains several subunits like alpha subunit , a beta subunit, as well as a gamma subunit.
An activated form of this G-protein has been released from the texture surface of the receptor as well as distinguished into its α- and β or γ subunits. According to Hasenoehrl et al. (2018), other types such as Gαq would be ultimately activated the PIPC (phospho-inositol phospholipase C) enzyme that hydrolyzed the (phosphatidylinositol-4,5- bisphosphate) turn into IP3 (inositol-1,4,5-triphosphate) as well as DAG (sn-1,2 diacylglycerol).
The IP3 has to be bound on an endoplasmic reticulum channel, which is made up of calcium that triggers the release of calcium ions on the cytosol. GPCR function has been associated with cell signaling of several environmental factors like ligand, metal, lights that help in the treatment of pain, cardiovascular disease as well as in cancer (Lobingier and von Zastrow, 2019).
3.3 Conclusion
Cell culture is a process by which cells are grown under the control environment that helps to elevate the other cell signaling process. Here in this assignment, GPCR or G protein-coupled receptor drugs are most diverse the largest as well as the group of membrane receptors present in eukaryotes.
It is also discussed in the assignment the LTK cell line is known as Leukocyte Tyrosine Kinase is a receptor. This assignment has been focused on giving a brief description of the process of cell culture as well as the effect on GPCR drugs on LTK cell lines. Therefore, this assignment is to provide a clear and accurate structure on the impact of GPCR drugs on LTK cell lines described by several tables and graphs.
Reference List
Hasenoehrl, C., Feuersinger, D., Sturm, E.M., Bärnthaler, T., Heitzer, E., Graf, R., Grill, M., Pichler, M., Beck, S., Butcher, L. and Thomas, D., (2018). ‘G protein‐coupled receptor GPR55 promotes colorectal cancer and has opposing effects to cannabinoid receptor 1’. International journal of cancer, 142(1), 121-132.
Insel, PA., Snead, A., Murray, F., and Zhang, L., (2012). ‘GPCR expression in tissues and cells: Are the optimal receptors being used as drug targets?’ Available from < https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3372817/> [18th November, 2020].
Kim, D. and Johnson, A.L., (2018). ‘Differentiation of the granulosa layer from hen prehierarchal follicles associated with follicle‐stimulating hormone receptor signaling’. Molecular reproduction and development, 85(8-9),729-737.
Lobingier, B.T. and von Zastrow, M., (2019). ‘When trafficking and signaling mix: How subcellular location shapes G protein‐coupled receptor activation of heterotrimeric G proteins’. Traffic, 20(2),130-136.
Mo, E.S., Cheng, Q., Reshetnyak, A.V., Schlessinger, J. and Nicoli, S., (2017). ‘Alk and Ltk ligands are essential for iridophore development in zebrafish mediated by the receptor tyrosine kinase Ltk’. Proceedings of the National Academy of Sciences, 114(45),12027-12032.
Okeke, K., Angers, S., Bouvier, M. and Michel, M.C., (2019). ‘Agonist‐induced desensitisation of β3‐adrenoceptors: Where, when, and how?’. British journal of pharmacology, 176(14),2539-2558.
Reshetnyak, A.V., Mohanty, J., Tomé, F., Puleo, D.E., Plotnikov, A.N., Ahmed, M., Kaur, N., Poliakov, A., Cinnaiyan, A.M., Lax, I. and Schlessinger, J., (2018). ‘Identification of a biologically active fragment of ALK and LTK-Ligand 2 (augmentor-α)’. Proceedings of the National Academy of Sciences, 115(33), 8340-8345.
Schiedel, A.C., Kose, M., Barreto, C., Bueschbell, B., Morra, G., Sensoy, O. and Moreira, I.S., (2018). ‘Prediction and targeting of interaction interfaces in G-protein coupled receptor oligomers’. Current Topics in Medicinal Chemistry, 18(8),714-746.
Know more about UniqueSubmission’s other writing services:
