Aim

The aim of this report is to evaluate the methodological bases of core research techniques, where the research questions are solved by comparing and contrasting. Further, the aim is to identify the common issues found in the research environment, so as to develop solutions for overcoming the challenges.

The objective of this report is to demonstrate the critical thinking and problem-solving skills in a research context.

The following are a set of hypothesis for this research:

• Can antibody titration data be useful?
• Is it important to analyze cell death in flow cytometry?
• The detection and exclusion of dead cells is essential in flow cytometry.
• Is it possible to determine the presence of non-viable cells?
• It is important to have the compensation controls.
• Is FlowJo helpful for analyzing the data?
• Should such a standard curve be used for gene expression quantification?

For Protein Extraction

The materials used for western blots are as follows:

• Phospho-lysis buffer stored at -80⁰C
• Protease inhibitors (PI) stored at -20⁰C

For Protein Denaturation

• 4x Laemmli sample buffer (LSB) stored at room temperature
• Dithiothreitol (DTT) stored at 4⁰C
• Heat block

For Protein Separation by gel electrophoresis

• Mini-protean TGX precast gel (4-15%) stored at 4⁰C
• 10x premixed electrophoresis buffer (25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3 following dilution to 1x with water)
• Long tips
• Marker: precision plus protein dual color standard stored at -20⁰C

For transferring the protein from the gel to the membrane  

• Pre-wetted Trans-Blot Turbo transfer packs (include filter papers, buffer and PVDF membranes, 1704156)
• Trans-Blot Turbo rapid western Blotting Transfer system

For incubation with antibodies

• Blocking Buffer stored at 40C. Dilute 1:1 in PBS
• Primary and secondary antibodies stored at -20⁰C
• PBS and Tween
• PBST = 500ml PBS + 500 µl Tween 20

Materials for qPCR

1. RNA extraction kit (Bioline) which is stored at a room temperature.
2. Molecular biology grade ethanol (100%).
3. Nuclease-free molecular biology grade water
4. RNase Away wipes

For cell culture

• Phorbol 12-myristate 13-acetate (PMA): Used to differentiate THP1 monocytes into macrophages.  
• Brefeldin A: inhibits intracellular transport from the endoplasmic reticulum to the Golgi complex so cytokines produced during activation are retained inside the cells.
• Lipopolysaccharides (LPS): Is a potent activator of monocytes and macrophages.

For staining cells and preparing FMOs

1. LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit −Cytofix/Cytoperm
2. FACS wash, perm wash and PBS
3. Antibodies
4. Cells to be stained

For Preparation of compensation controls for LIVE/DEAD: 1 tube

• ArC ™ Amine Reactive Compensation Bead Kit  
• LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit

For Preparation of compensation controls for all fluorophores (number of tubes =5)  

1. UltraComp eBeads™ Compensation Beads  
2. Antibodies  

• Antibodies used (state supplier, species, clones and catalogue number):

The antibodies used are 2ul IL-8 FITC, 5ul TNF BV421and 5ul IL-1β AF647, among which 2ul IL-8 FITC is used.

• Controls:

The controls are:

• Compensation controls
• FMO controls  
• How the images were generated and analyzed:

The images were generated by using Odyssey Imaging System (Li-Cor Biosciences) with the help of Image Studio (Ver 5.2) software. This software is utilized for capturing and exporting of data to TIF format.

• Gene accession numbers

low R2 value (<0.99) for a standard curve is used for gene accessing. Totally 10 reactions are accessed in the tables named, Plate 1: GAPDH and TNF-α and Plate 2: IL-1β and IL-8.

• Controls

Here, the controls includes:

• Positive Controls
• Negative Controls
• How the data was acquired and analyzed:

The QPCR analysis using the CFX MAESTROTM Software is used for analyzing the data and to retrieve the results.

• Antibodies used:

The antibodies used are LD aqua stain, cytofix/cytoperm and intracellular stains.

• Controls:

The controls are:

• Compensation controls
• FMO controls  
• How the data was acquired and analyzed:

The data is analyzed with WECC using FlowJo (FlowJo LLC).

The Cell culture and Protein Extraction are performed from which the results show that, Plating of cells and addition of PMA, LPS treatment and collection of cells at different time points.

When the Aliquot proteins are stored at -20⁰C, for a long time, the frequent freezing and thawing cycles result in protein degradation. Thus, it must be avoided. On the other hand, denaturation of the protein samples after calculating the amount of 4x LSB that is required to add to the amount of DTT which is present, is done. Then, the Protein Separation by gel electrophoresis is performed. Next, the protein is transferred from the gel to the membrane. Later, the Protein is transferred to the membrane. During protein transfer from a gel to the membrane, from the cassette the gel is removed and then to the membrane the protein is transferred. In case, if there is a need of re-wetting the PVDF membrane, make sure to dip it either into methanol or ethanol unless it becomes opaque uniformly. Only then, use the deionized water to wash it. This process is followed by incubation with blocking and probing of primary antibodies and secondary antibodies.

Objective

Therefore, the following steps comprises of blocking and probing of primary antibodies:

1. The membrane of 10ml with a diluted blocking buffer is incubated.
2. At RT, for an hour rock it.
3. From the tub, pour the blocking buffer into a 15ml tube.
4. The primary antibodies are added which are given by diluting it in 10 ml of blocking buffer, which contains 10 µl Tween.
5. At 4⁰C incubate the whole night by covering the tub with foil. This avoids evaporation of the staining solution.

Finally, the images are retrieved using Odyssey Imaging System (Li-Cor Biosciences) with the help of Image Studio (Ver 5.2) software. This software is utilized for capturing and exporting of data to TIF format.

                                       

                                               

                                                 

The above figure represents the result of both PI and PII. Here, the reagents are prepared with PCR mix of the following:

• 5 µl of 2x SYBR Green mix.
• Primer mix  
• 75 µl of F and R (300 nM).
• 75 µl of water.

The required materials are:

1. RNA extraction kit (Bioline) which is stored at a room temperature.
2. Molecular biology grade ethanol (100%).
3. Nuclease-free molecular biology grade water
4. RNase Away wipes

The required reagents are listed below:

• THP1 cells, human monocyte leukaemia cell line (catalogue # 88081201, Sigma-Aldrich) are separated as macrophage-like cells using phorbol 12-myristate 13-acetate (PMA) as macrophages mounts high responses to LPS When compared with monocytes.
• LPS (L3024, Sigma).
• PMA (P1585, sigma) stored at -20⁰
• RNA lysis Buffer (Bioline) and b-Mercapto-ethanol (b-ME).

The cell culture is performed first where on the first day, the cell are plated and PMA is added. The next day, LPS treatment is performed and also the cells are collected at various time points.

Then, RNA extraction takes place, where Spin column-based nucleic acid purification takes place. Where, Lysate filtration is done which discards cell debris along with denaturing the proteins. Then, RNA binding takes place followed by Desalting membrane; DNA digestion; Washing and drying Membrane; and Elution of RNA. Then, reverse transcription is done following by preparing a qPCR reaction mix. The preparation of qPCR master mix requires designing a layout of the samples that are used to assess. Later, positive control and negative controls are included. A table for 96 well PCR plate is tabulated, for working out on the total number of reactions (n), which will be assessed by the PCR. The tables are named as Plate 1: GAPDH and TNF-α and Plate 2: IL-1β and IL-8. To preparing a qPCR reaction mix, the following steps are included:

1. First, for Plate 1, the number of reactions (n) must be determined.
2. The volume required for every single PCR reaction component is calculated and mentioned.
3. The PCR MIX will be provided.
4. The required volumes will be combined.
5. Then for Plate 2, the number of reactions (n) must be determined.
6. The volume required for every single PCR reaction component is calculated and mentioned.
7. The PCR MIX will be provided.
8. The required volumes will be combined.

Totally, 20 µl of the PCR master mix is prepared. Finally, set up of the PCR takes place. Then, the QPCR analysis using the CFX MAESTROTM Software is used to retrieve the results.

In flow Cytometry, initially cell culture is performed at the point where the cells are stained and the FMOs are prepared, the cell could be fixed with the use of 200uL of cytofix for 20 min @ 4°C, only when the samples are not obtained on the exact same day.  Then, the cells must be washed once fixation takes place with 1mL FACS wash @ 1500 rpm 5 min, which is followed by a resuspension in 400 µL FACS wash. Make a point that the FSC and SSC voltages required for the beads are noted. The detector voltages of FSC and SSC can be changed, as various beads for the compensation controls are acquired. Next, the compensation controls can be recorded. With the help of BD FACSDiva (BD Biosciences), the data is acquired and recorded. Where, the instructions are set, created and the compensation is calculated.

Experimental design and materials

                                                 

Based on the core research techniques the methodologies are evaluated. The research questions are resolved on the basis of comparison and contrasting. In the research environment, the common issues are identified, for developing solutions so as to overcome the related challenges. On the other hand, the objective is met, where with respect to a research context, it demonstrates the critical thinking and problem-solving skills.  

In Western blot, when Aliquot proteins are stored at -20⁰C, for a long time, the frequent freezing and thawing cycles result in protein degradation. Thus, it must be avoided.  

Then, if a PVDF membrane needs re-wetting, ensure to dip it either into methanol or ethanol unless it becomes opaque uniformly. Later, use the deionized water to wash it.  

In PCR, Lysate filtration is performed. This steps ensures to discard the cell debris along with denaturing the proteins. Then, RNA binding takes place followed by Desalting membrane; DNA digestion; Washing and drying Membrane; and Elution of RNA.

In flow Cytometry, initially cell culture is performed at the point of staining cells and preparing FMOs, the cell could be fixed with the use of 200uL of cytofix for 20 min @ 4°C, only when the samples are not obtained on the exact same day.  Then, the cells must be washed once fixation takes place with 1mL FACS wash @ 1500 rpm 5 min, which is followed by a resuspension in 400 µL FACS wash. Make a point that the FSC and SSC voltages required for the beads are noted. The detector voltages of FSC and SSC can be changed, as various beads for the compensation controls are acquired. Next, the compensation controls can be recorded.

The methods used in this research work includes- Cell culture; wet or semi-dry methods of protein transfer from the polyacrylamide gel to the membrane; Probing for housekeeping gene;  BCA Assay (measuring protein concentration before loading to gel) and Ponceau S staining/ Coomassie is used for calculating the total protein in the lane. At last, for Western Blots, the images are acquired from the Odyssey Imaging System (Li-Cor Biosciences) of Image Studio (Ver 5.2) software which is utilized for capturing and exporting the data to TIF format. For flow cytometry, the data is analyzed with WECC using FlowJo (FlowJo LLC). For PCR, the QPCR analysis using the CFX MAESTROTM Software is utilized for analyzing the data and for retrieving the required results.