Lab Report

Published: 2021-06-11 06:25:03
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Lab Report Friend Leukemic Cell differentiation and Jurkat cell stimulation Anuradha Pullakhandam,22092148, University of Western Australia1. INTRODUCTION (i) General features of erythropoiesis with regard to changes in the pattern of gene expression and morphology, which accompany red blood cell differentiation: Erythropoiesis is a process of synthesizing the new and matured red blood cells through differentiation of the hemocytoblast (haemopoietic stem cells-HSC)1,2. These self- renewing, pluripotent stems cells proliferate and differentiate into another HSC and a committed progenitor, which can further differentiate into a specific cell lineage forming erythrocytes1,2. Red blood cells carry oxygen to various organs in the body2. To maintain the oxygen levels during hypoxia and anaemia, erythropoietin binds to a cellular receptor (Epo receptor) activating signalling pathways for generating red blood cells1,2. Epo receptor dimerises and phosphorylates due to the binding, which further activates JAK2 and STAT5 consecutively. STAT5 then dissociates and migrates to the nucleus stimulating the transcription factors such as BCL-xL, BCL-3, C-MYC and C-FOS which results in the activation of gene expression for cell growth and apoptosis1. In addition to this there are other pathways such as PLC-?, RAS and ERK pathways that also activate the gene expression for cell proliferation and differentiation3,4. Due to the activation of gene expression a committed proerythroblast differentiates forming a basophilic erythroblast which synthesizes ribosomes2. Basophilic erythroblast is further differentiated into polychromatic erythroblast where haemoglobin is synthesised2. During this process of differentiation, the size of cell and nucleus decreases consecutively forming a cell with smaller nucleus known as orthochromatic erythroblast. Finally, with the loss of organelles and depletion of nucleus a reticulocyte is formed. These immature red cells lose organelle fragments forming a new and matured erythrocyte2.(ii) The general features of T-cell activation and the cell signalling processes that occur following cell stimulation with PHA and synthesis and secretion of IL-2: T cells are a type of lymphocytes which are also generated through a specific cell lineage1. These white blood cells are responsible for maintaining the immunity in the body. The process of synthesis is initiated by the stimulation of the T-cell receptor complex (TCR/CD3). PHA stimulates TCR by binding to the complex and this in turn activates tyrosine kinases such as Lck, ZAP70 and Syk and these kinases phosphorylate and activate PLC? subsequently5. Activation of PLC? leads to the production of IP3(Inositol-1,4,5-triphosphate) and DAG(diacylglycerol) through hydrolysis of phosphatidylinositol-3-4-bisphosphate(PtdIns(3,4)P2), IP3 releases calcium that is deposited in the cells6. As a result, intracellular calcium concentration is increased and consequently it results in activation of several transcription factors such as NF-AT, CREB and NF-kB which leads to cell proliferation, differentiation of apoptosis5,6. Activation of the transcription factor NF-kB and co stimulation of CD28 leads to initiation of T cells which synthesizes and secretion of IL25.2. RESULTS (i) Friend Leukemic Cells a. Haemoglobin standard curve and the absorbance values of the samples measured. Table1: The table below shows the absorbance readings of the Standards in rows 1 and 2. Row 3 shows absorbance reading of 1/2 dilution of the untreated sample from A-C and DMSO treated sample from D-F. Row 4 shows 1/4th dilution of the untreated from rows A-C and DMSO treated sample from D-F and row 5 indicates the absorbance readings of 1/8th diluted untreated samples from A-C and DMSO treated from D-F and finally, row 6 depicts the absorbance readings of 1/16th diluted untreated samples from A-C and DMSO treated from D-F. 1 2 3 4 5 6 Standard 1 Standard 2 1/2 1/4 1/8 1/16 A 0.149 0.149 0.22 0.167 0.166 0.152 B 0.175 0.168 0.209 0.166 0.152 0.144 C 0.198 0.188 0.207 0.168 0.161 0.145 D 0.242 0.235 0.334 0.252 0.204 0.168 E 0.304 0.271 0.809 0.317 0.22 0.178 F 0.557 0.29 0.218 0.184Summary of the results:i.) Table 2: showing the summary of the results of untreated and DMSO treated Hb content/cell and Hb content /differentiated cell from Haemoglobin assay % positive Hb cells Hb content/cell (pg/cell)(mean ± std deviation) Hb content/differentiated cell (pg/cell)(mean ± std deviation)Untreated Culture 0 2.13 ± 2.3 * 10-1 ———DMSO-treated culture 52 8.79 ± 1.16 16.8974 ± 2.23533Individual absorbance measurements for the XTT assay: 0 hour: 485nm 1 2 3 4 C 0.238 0.338 0.331 0.336 D 0.401 0.24 0.257 0.2480 hour: 650nm 1 2 3 4 C 0.262 0.324 0.311 0.325 D 0.45 0.242 0.257 0.24 1 hour:485nm 1 2 3 4 C 0.329 1.741 1.689 1.024 D 0.511 0.678 0.842 0.5641 hour: 650nm 1 2 3 4 C 0.289 0.343 0.332 0.347 D 0.502 0.278 0.301 0.248 Summary of the Results:ii. Table 7: showing the summary of results of untreated and DMSO treated culture using XTT activity Untreated FLC culture DMSO treated culture% positive Hb cells XTT activity(?A485 / 3 * 105 cells/hr)(mean ± std deviation) % positive Hb cells XTT activity(?A485 / 3 * 105 cells/hr)(mean ± std deviation) 0 1.065 ± 0.401658 52 0.18668 ± 0.060955b. Comparisons of data between treated and untreated cells i. Comparison of Hb content data activity in pg/cell between Untreated and DMSO-treated cultures:The Hb content data activity for untreated culture is 2.13 ± 2.3 * 10-1 pg/cell and DMSO – treated cultures is 8.79 ± 1.16 pg/cell. The results of mean and standard deviation indicate that there is significant increase in the haemoglobin content in DMSO -treated cultures when compared to untreated samples. The p valued calculated using T. test is 0.0006, which is significantly less than 0.05. Therefore, from these two results it can be confirmed that there is a significant difference between the Untreated ad DMSO-treated cultures.ii. Comparison of XTT activity (?A485/3×105 cells/hr) between Untreated & DMSO-treated cultures: The XTT activity (?A485/3×105 cells/hr) of Untreated culture is 1.065 ± 0.401658 and DMSO – treated culture is 0.18668 ± 0.060955 show that there is decreased activity in the DMSO – treated samples in comparison with the Untreated culture. This can be confirmed with the p value obtained from the T.test, which is 0.04 which is less than 0.05. These results indicate that there is significant difference between the Untreated and DMSO-treated cultures.(ii) Jurkat T-Cells a. (i) Four time course graphs of changes in intracellular Ca2+ concentration in the samples measured: (ii) Images of the pre- and post-transfer gels along with the post-transfer blot and the image of the blot following the detection procedure: SDS gel electrophoresis/western blotting has been performed with the addition of IkB? (different stimulations), prestained, unstained and +ve control. Pre and post transfer images exhibited bands at around 70kDa in rows 4-7(PMA +A23187, PHA+PMA, PHA and control). Positive control(row1) also exhibits bands around 70kDa in pre and post transfer images. Presence of bands in pre-gel image show that there is protein in the sample. Bands in the western blot indicate that there is binding of the protein with the antibodies11.Western blot images show that there is abundant amount of protein present in the samples, which indicates that the protein has been synthesized. However, patchy and uneven lines are observed in the prestained sample, this might be because of improper transfer of the standards on to the membrane or it might be because of the accidental heating of the prestained standard which might have denatured the proteins7,11. Even though the bands are clearly visible in pre – gel image, they are fainter than the post transfer image. The Lanes 2(unstained standard) and 3 (prestained standard) are alike in the post transfer gel image, this is due to the error in pipetting while loading the gels. Some of my sample from lane 3 has accidently mixed with lane 2. (iii) IL-2 standard curve with trendline fitted showing the equation of the trendline and the R2 value: (iv) Absorbance measurements for samples in the IL-2 assay: Table 8: the table below shows the absorbance readings at 450nm. lanes A and B indicate the absorbance reading of the standards, C1, C2 and C3 are the absorbance readings of PMA +A231487, C4, C5 and C6 depict the reading of PHA + PMA, D1, D2 and D3 are the readings of PHA and finally, D4, D5 and D6 show reading of a control sample. 1 2 3 4 5 6 7 8 9A 0.185 0.182 0.182 0.189 0.2 0.251 0.313 0.424 0.657B 0.177 0.185 0.181 0.191 0.209 0.238 0.307 0.417 0.615C 1.128 1.121 0.973 0.332 0.328 0.32 D 0.221 0.219 0.207 0.17 0.172 0.165 Summary of the results:(v) Table 9: Data from intracellular Ca2+ concentration measurements, Western Blot analysis and IL-2 measurements are listed in the table below: Sample ?[Ca2+]max (nM)(Mean ± S.D.) PhosphoIkB?(-,+,++)* [IL-2] (pg/ml)(Mean ± S.D.)a. Control 45.44535 ± 26.88284 – -b. PHA 299.6715 ± 32.97616 + -c. PHA + PMA 298.6559 ± 28.3574 + -d. PMA + A23187 396.1824 ± 77.18289 + -b. Comparisons of data between stimulated and unstimulated cells and between the different cell stimulations: By analysing the data obtained from the calcium assay(?[Ca2+]max (nM)) it can be implied that there is a significant difference between the PMA+A23187, PHA + PMA, PHA and the control. Average and standard deviation values of the control (45.44535 ± 26.88284nM) are significantly less than the values obtained for PMA +A23187(396.1824 ± 77.18289nM), PHA + PMA (298.6559 ± 28.3574nM), PHA (299.6715 ± 32.97616nM). This is also confirmed from the p value obtained through the T.test, the p value of control and PMA+A23187 is 0.0017, the p value of control and PHA+PMA is 0.0003 and the value for control and PHA is 0.0004 which show significant difference as standard p

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