Flow Cytometry ExperimentsReport-By Drashti Shah, Roll: 111.

Propidium Iodide Uptake:Aim: To analyse cell viability in A549 cell line after treating them with ethanol using Propidium Iodide dye.Principle: In this experiment, we want to differentiate between live and dead cells. For this purpose we use Propidium iodide, which is a membrane impermeable dye. Live cells have semi-permeable membranes which allow only selective molecules to pass through via pores in the membrane. But, dead cells lose their ability to block such foreign substances since their membrane becomes permeable. Hence, after treating the cells with ethanol we check for cell viability using Propidium iodide. The PI dye excitation is at 488nm and its emission wavelength is at 617nm.

Procedure:A fully confluent flask was trypsinized and cells were counted using a haemocytometer under an inverted phase contrast microscope.106 cells were seeded in each well. 1 well was kept for control in which no treatment was given and the media was changed if required.Remaining wells were treated with different concentrations of ethanol being 5%, 10%, 25% and 50%. Then the plates were incubated at 37 ?C and 5% CO2 overnight.

Next, the cells were harvested at 8000 rpm for 5 minutes for flow Cytometry analysis.Trypsinization of cells was carried out by trypsin-EDTA and complete media was added.The pellets were resuspended in 100 microliter PBS.20 microliter PI dye was added and incubated for half hour.Samples were analysed in flow cytometer using dot plot (FSC and SSC) and histogram.Results:(Histogram)-76835027305000571500(Result Data)00(Result Data)-7620040894000 Discussion:In the histograms plotted, M2 is the gated population which show the cells with PI dye, meaning that these are dead cells. M1 cells are the viable cells.

As we can see, the dead cell number increases steadily from 5% up to 50%. There is one discrepancy in the result meaning that the dead cell number in control sample is a bit more than that in 5% sample. This can be due to pipetting errors which led to more cell death in control sample.2. Nanoparticle Uptake (TiO2):Aim: To analyse the cellular uptake in A549 cell line using TiO2 nanoparticles.Principle: Nanoparticle based drug delivery is a very upcoming research and can prove to be very effective if applied strategically.

For this purpose, we need to check whether cells uptake these nanoparticles or not. In this experiment, we check TiO2 nanoparticle uptake. There can be a lot of reasons why the cells are unable to uptake the nanoparticles.

Sometimes, the nanoparticles agglomerate easily and hence cells cannot take it in. Different media has different effects on the experiment so using appropriate media is also important. Protocol:A fully confluent flask was trypsinized and cells were counted using a haemocytometer under an inverted phase contrast microscope.106 cells were seeded in each well.

2 wells were kept for control in which no treatment was given and the media was changed if required (DMEM media was used)Treatment of TiO2 nanoparticles was given with different concentrations of 5, 10 and 25 micrograms.Then the plates were incubated at 37 ?C and 5% CO2 overnight.Next, the cells were harvested at 8000 rpm for 5 minutes for flow Cytometry analysis.Trypsinization of cells was carried out by trypsin-EDTA and complete media was added.The pellets were resuspended in 300 microliter PBS.The sample was analysed in flow cytometer with dot plot and histogram.

Results:28638527368500(Dot plot)7810530099000-50800000Discussion:As cells uptake nanoparticles, their granularity increases. This leads to an increase in SSC parameter of the dot plot. When gated and put into histogram parameters, we find the number of cells which took in nanoparticles and which didn’t. In the table above, M2 is the gated population which is the desired population. As we can see, there is an increase of M2 from control to 5% but there is a decrease to 10% which can be due to pipetting or other errors. The nanoparticle uptake again increases in 25% which is in line with our theory.3.

ROS Formation:Aim:To analyse ROS generation in WRL cell line by TiO2 Treatment.Introduction:Reactive Oxygen Species (ROS) are chemically reactive species which contain oxygen. Free radicals are formed which is an atom or a group of atoms that have one or more number of unpaired electrons. They can have positive, negative or neutral charge. Various environmental stresses lead to over the top ROS production which then leads to oxidative damage and ends in cell death. Administering certain drugs can also cause ROS formation.

In this experiment, we use TiO2 nanoparticles to compare the ROS formation due to different concentrations. To check for ROS production, cell permeable DCF-DA dye is used. The non-fluorescent 2′,7′-dichlorodihydrofluorescein diacetate fluorescein gets oxidized and converts into fluorescent 2′,7′-dichlorofluorescein by presence of intracellular ROS.

Protocol:Seed 1.5 lakh cells/ml in 6 well plate from a confluent flask of WRL cell line in MEM media.Prepare different concentration of 10, 25, 50 and 100 µg/ml of TiO2 nanoparticle treatment.Give gold nanoparticle treatment to the cells and incubate it for 6 hrs.Add 20uM of DCF-DA dye in 1ml and then incubate at 37o C for 30 minutes.Centrifuge the cells at 250 g for 10 minutes.

Discard the supernatant and resuspend the cell pellet in 1 ml of PBS.Analyse the sample in flow cytometer.Result:14033503429000 400050220980001560830180340000666750102001439545239649000Discussion:In the results, M1 is the gated population, meaning this is the population which shows fluorescence due to ROS formation. The 25 microgram/ml concentration shows maximum ROS formation. The fall in 50 microgram/ml can be due to pipetting or some other human errors.

4. Cell Cycle:Aim:To analyse different stages of cell cycle using Propidium iodide dye.Introduction:Cell cycle consists the processes starting from DNA material replication in the cell up to the division of cells into two daughter cells. The cells cycle is divided into multiple phases and according to activities which take place, the flow cytometer differentiates between the different phases. For example, in the S phase, the cell duplicates its DNA material which will in turn increase the cell size and this will be reflected in the FSC of the dot plot. Similarly, other phases of the cell cycle are also observed.

The PI dye binds to the DNA of the lysed cells and hence fluorescence is analysed.The PI dye excitation is at 488nm and its emission wavelength is at 617nm.Protocol:Seed 2 lakh cells per well in a 6 well plate, overnight.Nanoparticles (TiO2) treatment was given to cells for 8 hours. Following concentrations – 5 µg/ml, 10 µg/ml, 25 µg/ml and 50 µg/ml.Harvest the cells using trypsin EDTA and wash them with 1x PBS.

Fix them in 70% ethanol prepared in 1x PBS. Store the cells at -200 for 30 minutes.Centrifuge the cells at 1200 rpm for 10 minutes. Add 0.2% Triton X to the cell pellet and incubate the cells at 40C for 1 hour.Centrifuge at 1200 rpm for 10 minutes.

Resuspend the cell pellet in 1ml PBS with 20µl RNAse (10mg/ml), keep them at 370C for 30 minutes.Again centrifuge at 1200 rpm for 10 minutes.Add PI and incubate the cells for 30 minutes at 40C.

Transfer the samples in the flow tubes and label them respectively.Observe under flow cytometer.99695021083900Results:73025000073025027495500Discussion:In the graphs, the M1 gate is of the G1 phase, M2 gate is of the S phase and the M3 gate is of the G2 phase. The general trend observed is that the number of cells entering into G2 phase become lower as the concentration of TiO2 increases. At the 25µg, the cells are arrested at G1 phase and even the percentage of cells in S phase declines. This shows that the nanoparticles at 25µg cause enough damage to not let the cell cross the G1-S phase checkpoint. The only reason why there is a slight increase in the cell number in G2 phase in 50µg treatment is because of cell clumping or uneven cell distribution.

5. Immunophenotyping:Aim:To identify different T cells and B cells from the blood with the help of various surface markers using flow cytometer.Introduction:Immunophenotyping is a term applied to the technique of identifying the specific lineage of cells through the use of antibodies that detect antigens or markers on the cell.

The markers can be expressed on the cell surface, in the cytoplasm or the nucleus of the cells. A given cell population can be identified and quantified through unique cell markers using different antibodies coupled with fluorochromes. Although immunophenotyping provides much useful information, it is a technique that is best used to examine specific cell populations.Protocol:PBMC Isolation-Collect 4 ml of blood in the EDTA/Heparin coated collection tube, using sterile syringe and needle. Mix immediately by inverting or vigorously shaking the tube for EDTA/ Heparin to be uniformly distributed. Dilute the blood by adding 4 ml of diluent buffer. Take 3 ml of HiSep LSM 1077 in a new 15 ml centrifuge tube. Carefully layer the diluted blood sample (4 ml) onto the Hisep solution (do not mix the sample while layering)Centrifuge at 400 g with brake off, at room temperature (15?25°C) for 30 minutes.

Centrifugation should sediment erythrocytes and polynuclear leukocytes and band mononuclear lymphocytes above HiSep LSM 1077. Using pipette carefully aspirate the interface containing mononuclear cells and transfer it to a clean centrifuge tube.Add equal volume of isotonic phosphate buffered saline to mononuclear cells and mix by gentle aspiration. Centrifuge at 160? 260 g with brake off, at room temperature (15?25°C) for 10 minutes.

This washing step with isotonic phosphate buffered saline removes HiSep LSM and reduces the number of platelets. Discard the supernatant and suspend pellet in 500 µl of isotonic buffer or mediumSample Preparation:Take 100ul of lymphocytes solution and add 20ul of antibody cocktail to the tube.Vortex the tube gently.Incubate in dark at room temperature for 10-15 minutes.

Add 2ml BDFACS lyse (1X).Vortex and incubate the tube in dark at room temperature for 10-12 minutes.Centrifuge the cells at 200-300 g for 5minutes.Discard the supernatant.After resuspending, wash the cells with sheath fluid, vortex gently and repeat centrifugation step.Resuspend the cells in 0.

5 ml sheath fluid or PBS with 2% paraformaldehyde.Acquire the sample using flow cytometer.50165041656000Results:19812046101000Discussion:From the above dot plots, we can clearly see the distribution of different T cells and B cells.

All the four graphs show clear differentiation between T cells and B cells.In the second graph having FL2 and FL3, it represents the CD4 and CD3. We know that CD3 represents T cells and CD4 represents T helper cells. So, in second graph we can see that UR (upper right) are T helper cells which are 17%.

The LR shows T cytotoxic cells which are 28%.In third graph having FL3 and FL1, the UR shows T cytotoxic cells which are 17% and LR are T helper cells which are 45%. We can clearly compare both the graphs the graphs and we can observe that cells are nearly similar in concentrations.In the fourth graph having CD4 and CD8, the cells in LR shows T cytotoxic cells and cells in UL shows T helper cells. They are 18% and 33% respectively. After comparing third and fourth graphs, here the T cytotoxic cells are similar in concentrations but the T helper cells are little bit more in third graph than fourth graph.

The reason could be due to the compensation as properly compensation would not been occurred which resulted in difference in concentrations. 6. Apoptosis:Aim: To analyse the phenomenon of apoptosis in the cells using FITC Annexin – V antibody and PI dye.Introduction:Apoptosis in cells in programmed cell death. It occurs due to external or internal stimuli as well as maintenance of tissue homeostasis, under pathological conditions, and in aging. The process is characterized by specific morphologic features, including loss of plasma membrane asymmetry and attachment, plasma membrane blebbing, condensation of the cytoplasm and nucleus, and internucleosomal cleavage of DNA. In apoptotic cells, the membrane phospholipid phosphatidylserine (PS) is translocated from the inner to the outer leaflet of the plasma membrane, thereby exposing PS to the external cellular environment.

Annexin V is a Ca2+-dependent phospholipid-binding protein with high affinity for PS, and binds to exposed apoptotic cell surface PS. 2–4 Annexin V can be conjugated to fluorochromes while retaining its high affinity for PS and serves as a sensitive probe for flow cytometric analysis of cells undergoing apoptosis. Since FITC tagged Annexin – V antibody analyzes the latest stages of apoptosis with loss of membrane integrity, a viable dye (PI dye) is used to analyze the earlier stages of apoptosis including pore formation, leading to entry of the dye. Cells that stain positive for FITC Annexin – V and negative for PI are undergoing apoptosis.

Cells that stain positive for both are either undergoing necrosis or already dead. Cells that stain negative for both are alive and not undergoing measurable apoptosis. Cells that stain negative for Annexin – V and positive for PI are in early stage of apoptosis.Protocol: A fully confluent flask was trypsinized and cells were counted using a haemocytometer under an inverted phase contrast microscope and 106 cells were seeded in each well.Prepare camtothecin stock solution (1mM in DMSO) as positive control.

Prepare its working solution of 4-6 µM.Treat the cells with camtothecin working solution and incubate them for 4 to 6 hrs at 37o C.Wash the cells twice with cold PBS and then resuspend in 1x binding buffer at concentration 1 x 106 cells/ml.Transfer 100ul of this cell suspension and add 5 ul of FITC and 5ul of PI.

Gently vortex the cells and incubate them in dark for 15 minutes at room temperature.Add 400ul of 1X binding buffer to each tube.Acquire the cell sample by flow cytometry within an hour.

Results:1022350-6908800013525501841500305879501016000 Discussion:In above shown results FL2 filter is for PI dye and FL1 filter is for FITC.The upper left quadrant represents the cells undergoing apoptosis. The upper right quadrant represents necrosis or dead cells. The lower left quadrant represent live cells and the lower right quadrant represents the cells at early stage of apoptosis. As the concentration of camtothecin treatment increases, percentage of necrotic cells increases.