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Lab 2

Isolation of mononuclear cells from peripheral blood and separation into

subpopulations

Supervisors: Sissela Broos sissela.broos@immun.lth.se tel: 222 96 78 Niclas Olsson niclas.olsson@immun.lth.se tel: 222 94 13

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Flowchart

Filter material

Ficoll-paque

Lymphocytes and monocytes (PBMC)

Binding to plastics

Rosetting

Adherent cells (monocytes) T-cells fraction

B-cell fraction (incl. monocytes)

Aim: To gain an insight into the purification of lymphocytes and analysis of antigen expression on different cell populations.

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Introduction Blood contain different types of cells, for example red blood cells (erythrocytes), which transport oxygen to all tissues in the body and white blood cells (leukocytes), which are part of the immune system. In this lab we are going to look more closely at some of the white blood cell populations. We will do so using flow cytometry which is a common technique for analysis of cells in clinical laboratories. In order to perform this analysis, we must first separate different types of leukocytes from each other by using the unique characteristics of the cells. We will isolate T-lymphocytes, monocytes and B-lymphocytes. The aim of the lab is to give an insight into different cell separation methods, as well as how to analyze cells using flow cytometry. We will use a so called ”buffy coat”, which is the remaining part when red blood cells are separated from the blood plasma in human blood. The “buffy” is obtained from the blood bank at Lund University Hospital. During day 1, we will carry out inital preparations to separate leukocytes from blood and subsequently isolate the different cell populations (monocytes, B-cells and T-cells). First, we will dispose off the red blood cells by performing a density gradient centrifugation on Ficoll-Paque. The PBMCs (peripheral blood mononuclear cells) will form a layer in the tube that will be further purified. Monocytes will be isolated by utilizing their ability to bind to plastic surfaces. T-lymphocytes will be separated using activated erythrocytes from sheep, which bind to a molecule on the surface of T-cells. The T-cell depleted fraction will then contain both B-cells and monocytes. The different cell populations will finally be studied using flow cytometry on day 2.

All donors are tested before becoming approved blood donors, but there is still a risk that the material may be contagious when we receive it, so it is vital to work very carefully.

Important to also bear in mind when working with cells:

1. Always keep the cell suspensions in tubes on ice. 2. Resuspend a cell pellet in a small volume of liquid first and then add more liquid.

Otherwise the cells will form “clumps”. 3. Keep the lymphocytes at the right pH (6,8-7,4), even though they endure low pH better

than high pH. 4. Work gently with the cells, they are small living creatures. Do not pipette quickly.

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Isolation of mononuclear leukocytes In blood transfusions it is important that the blood does not contain any white blood cells. These are separated from the blood by filtration and the collected cells are referred to as filter material (formerly known as buffy coat, which is separated by centrifugation). From this you can obtain mononuclear cells using a density gradient centrifugation on Ficoll-Paque (density = 1.078). Differencies in density will separate lymphocytes from other blood cells. After the centrifugation three or four fractions are visible: Counting cells in a Bürker chamber

Centrifugation Plasma

Lymfoc., monocyt. & tromb. δ < 1.078

Ficoll-Paque

Granulocytes δ > 1.078 Aggregated erythrocytes δ > 1.078

Blood sample

Ficoll-Paque

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The proportion of viable cells can be determined by staining the cells with trypan blue. Viable cells will not become stained while dead cells will become blue. The cells have to be counted within 3-4 minutes or the viable cells will also take up the colour. Bürker chambers and cover slips should be properly cleaned in water and alcohol. The cover slip is attached to the glass by humidifying the borders of the glass. Dilute the cells in trypan blue and let the cell suspension slip in under the cover, into one of the chambers. Abundant cell suspension should be dried off using a Kleenex. Each field in the counting chamber is divided into 9 squares separated with triple lines and such a field (A-square) contain 0,1 µl. The A-square is further divided into 16 parts, B-squares (see figure below). When counting cells in an A-square, all cells that touch the right and the upper border should be included while cells on the left and bottom line should be excluded. Count at least a hundred cells (sometimes you may have to count more than one A-square) and use the average to determine the concentration of cells. Amount of viable cells/ml=viable cells in one A-square x 104 x dilution factor

B-square

A-square

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Flow cytometry Flow cytometry is a mean of measuring certain physical and chemical characteristics of cells or particles as they travel in suspension one by one past a sensing point. The modern flow cytometer consists of a light source, collection optics, electronics and a computer to translate signals to data. In most modern cytometers the light source of choice is a laser, which emits coherent light at a specified wavelength. Scattered and emitted fluorescent light is collected by two lenses (one set in front of the light source and one set at right angles) and by a series of optics, beam splitters and filters to allow specific bands of fluorescence to be measured. Physical characteristics such as cell size, shape, internal complexity and any cell component or function that can be detected by a fluorescent compound can be examined. So the applications of Flow Cytometry are numerous, and this has led to the widespread use of these instruments in the biological and medical fields.

The term "Flow Cytometry" derives from the measurement (meter) of single cells (cyto) as they flow past a series of detectors. The acronym FACS (Fluorescence Activated Cell Sorting) and Flow Cytometry are used interchangeably. The fundamental concept is that cells flow one at a time through a region of interrogation where multiple biophysical properties of each cell can be measured at rates of over 1000 cells per second. These biophysical properties are then correlated with biological and biochemical properties of interest. The high through-put of cells allows for rare cells, which may have inherent or inducible differences, to be easily detected and identified from the remainder of the cell population.

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In order to make the measurement of biological/biochemical properties of interest easier, the cells are usually stained with fluorescent dyes, which bind specifically to cellular constituents. The dyes are excited by the laser beam, and emit light at a longer wavelength. Photo Multiplicator Tubes (PMT) detects the emitted light as an analogue electronic signal, which subsequently is converted to digital information to be analyzed in a computer. Three types of data are generated: Forward scatter (FSC) Approximate cell size Side/orthogonal scatter (SSC) Cell complexity or granularity Fluorescence Fluorescent labeling is used to investigate cell structure and function

Scatter Forward and side scatter are used for preliminary identification of cells. In a peripheral blood sample, lymphocyte, monocyte and granulocyte populations can be defined on the basis of forward and side scatter. Forward and side scatter are also used to exclude debris and dead cells. Fluorescence Labeling cells with fluorescent dyes allows investigation of structure and function. Fluorescence intensities are typically measured at several different wavelengths simultaneously for each cell. Fluorescent probes are used to report the quantities of specific components of the cells. Fluorescent antibodies are often used to report the densities of specific surface receptors, and thus to distinguish subpopulations of differentiated cell types, including cells expressing a transgene. By making them fluorescent, the binding of viruses or hormones to surface receptors can be measured. Intracellular components can also be reported by fluorescent probes, including total DNA/cell (allowing cell cycle analysis), newly synthesized DNA, specific nucleotide sequences in DNA or mRNA, filamentous actin, and any structure for which an antibody is available. Flow cytometry can also monitor rapid changes in intracellular free calcium, membrane potential, pH, or free fatty acids. Immunofluorescence, the most widely used application, involves the staining of cells with antibodies conjugated to fluorescent dyes such as fluorescein (FITC) and phycoerythrin (PE). This method is often used to label molecules on the cell surface, but antibodies can also be directed at intracellular targets in the cytoplasm. In direct staining the antibody is directly conjugated to a fluorescent dye (e.g. anti-CD4 PE). Cells are stained in one step. In indirect staining the primary antibody is not labeled. A second antibody conjugated to a fluorescent dye, and specific for the first antibody is added. For example, if the anti-CD4 antibody was a mouse IgG then the second antibody could be a rat antibody raised against mouse IgG.

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Analysis The ability of Flow Cytometers to evaluate cells at an extremely rapid rate (e.g. up to 20,000 events per second) makes this technology ideally suited for the reliable and accurate quantitative analysis of selected physical properties of cells of interest. The sensitivity of these instruments for detecting the presence of molecules expressed at low levels is impressive; given high quality cell preparations and reagents, as few as 500 molecules per cell may be detected. At the Department of Immunotechnology the Becton-Dickinson FACS Canto II will be used for analysis. The BD FACSCanto system is built with blue (488 nm, air-cooled, 20 mW solid state) and red (633 nm, 17 mW HeNe) excitation sources. The laser beams are then routed via fiber optics to the beam-shaping prisms where two lasers are projected onto separate spots in the flow cell. Here, the particles intercept with the laser excitation beams and optical signals are collected at the backside of the flow cell with a gel-coupled collection lens. The system has 6 different colour detectors and several different dyes can thereby be used. In addition the system has a very fast acquisition rates (up 10,000 events per sec).

Cell sorting One of the properties of the larger flow cytometers is the ability to electronically deflect cells with preset, defined properties into a separate collection tube. In these instruments the fluidics hydrodynamically focuses the cell stream to within an uncertainty of a small fraction of a cell diameter and break the stream into uniform-sized droplets to separate individual cells. The electronics quantitate the faint flashes of scattered and fluorescent light, and, under computer control, electrically charge droplets containing cells of interest so that they can be deflected into a separate test tube or culture wells. For cell purification, flow cytometry is especially well suited for applications requiring high purity. Because multiple fluorochromes (e.g. up to five distinct fluorescent probes reacting with different cell associated molecules) can be assessed simultaneously, cell sorting by flow cytometry can separate complex mixtures of cells on the basis of multiple marker expression. At the Department of Immunotechnology we use a Becton-Dickinson FACS Aria for cell sorting. This Aria is a 9 detectors instrument with 3 lasers and digital acquisition rates of up to 70,000 events/second. The bitmapped sorting capability of the Aria digital electronics allows the operator to set up sophisticated sort decisions using logical gates combined with the Boolean operators AND, OR, and NOT. The Aria can also be used for multicolor assays with too many colors (nine) for the other instrument.

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Important! The laboratory manual is only a guide. Some changes may occur during the lab and these must be documented and described in the lab report. DAY 1: 1. Fill a T75:a with 250 ml PBS. Cut the end of the filter tubing that is least

blood filled and connect it to the vacuum tubing. Cut the other side and place it in the T75 with PBS. Turn on the vacuum and wait until all PBS has gone through the filter. Turn the vacuum off.

2. Disconnect the bucket from the vacuum tubing and carefully put it in the

LAF bench. Carefully swirl the bucket and remove the top. Transfer the blood solution into 6 x 50 ml Falcon tubes using a 50 ml stripette. Fill up the last tube with PBS to balance in the centrifuge.

3. Centrifuge the tubes at 1800 rpm for 5 min (room temp.) 4. Aspirate the supernatant and leave about 10 ml in each tube. Pool the

solution into two of the tubes and add PBS for a total volume of 75 ml. 5. Place 15 ml of Ficoll Paque into three 50 ml tubes/group. 6. Carefully transfer 25 ml of the diluted filter material ONTO the Ficoll Paque

so that they form two separate layers. 7. Centrifuge in room temperature at 1800 rpm for 20 min without breaks. 8. The leukocytes can now be collected at the interphase between the Ficoll

Paque and the plasma (se figure at page 4). Transfer them carefully using a 10 ml pipette or a pasteur pipette into four new 50 ml tubes. Try to avoid aspirating the plasma and Ficoll Paque.

9. Dilute the cell suspensions with PBS up to 50 ml in each tube.

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10. Centrifuge 1250 rpm for 10 min (room temperature). 11. Aspirate the supernatants and be careful not to lose the cells. Resuspend the

pellet BEFORE PBS is added. 12. Resuspend the cells in 10 ml cold PBS and pool the cells into one tube.

Always keep the cells on ice after this point. 13. Centrifuge the cells at 900 rpm for 10 min (4°C). The leukocytes will now

form a pellet while the trombocytes will stay in the supernatant. 14. Carefully aspirate the supernatant (the pellet is loose!!!). Resuspend the cells

in 10 ml PBS, take out a sample and count the cells (dilute 50 times before counting) (see page 5). Repeat the washing step (number 10) while you are counting.

15. Resuspend the cells in cell culture medium (R5) to a cell concentration of 10

x 106 cells/ml. 16. Take out 3 times 50µl samples and transfer them to 3 different 5 ml tubes

(”FACS tubes”) (=0,5 x 106 cells). Mark them with A1, A2 and A3 and the name of the group and keep them in the fridge before the analysis on day 2.

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Binding of adherent cells to plastic surfaces The cells (from step 12) that we have at this stage are lymphocytes and monocytes. Monocytes could be separated by plastic adhesion, since they adhere to plastic surfaces and lymphocytes do not. Take ~ 75 x 106 cells (<1/3 of all cells) to this experiment. The rest of the cells are used for rosette formation (see next page).

1. Take ~ 75 x 106 cells (from step 12) and add R5 to a total volume of 15 ml. Transfer to a T75 flask.

2. Look at the cells in microscope. Incubate the cells at 37°C for 2-4 hours.

3. After 2 hours, take out the flasks and look at the cells in microscope. The

monocytes that have adhered to the plastic surface looks like ”flattened amoebas”. If you can not see this yet, incubate the cells for longer period of time and check them every half hour.

4. When there is a distinct carpet of cells in the bottom of the flask, remove all

the media and put it in the waste.

5. Wash away T and B cells by adding new R5 media (10 ml) and then aspirate it. Repeat once again.

6. Look at the flask in microscope, if there are still cells present that are not

bound to the surface, repeat step 5 until no unbound cells can be detected.

7. After the last wash, add 15 ml R5 to the flask in which you have monocytes. The monocytes are incubated at 37°C over night. The cells will then no longer be bound to the surface and can be used in another test (in our case we will stain them with fluorescence-labeled anti-monocytes-antibodies and analyze them using flow cytometry).

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Rosette formation of T-cells with red blood cells from sheep Erythrocytes from sheep (FRBK) have a natural affinity for human T lymphocytes (binds to a marker on T lymphocytes that is called CD2). This has for a long time been used for positive selection of T lymphocytes. When the surface of T lymphocytes are covered with erythrocytes small aggregates (rosettes) are formed that can easily be separated from the other lymphocytes by gradient centrifugation (Ficoll or Percoll). 1. The rest of the cellsuspension (~12.5 ml from step 15 above, 10*106

cells/ml) is divided into two 50 ml tubes. Resuspend a tube with FRBK (10%) by carefully tilting it upside down a couple of times. Add 10 ml of activated FRBK to each tube with cellsuspension. Keep Cold! Centrifuge 900 rpm for 3 min. Breaks off!!

2. Incubate on ice for 25 min. 3. Add 4 ml Ficoll (1.078 g/ml) to 4 different 15 ml tubes. Stop the

incubation above. Dissolve the pellet and transfer the suspension really gently ONTO the Ficoll so that 2 separate layers will form. Leave a little droplet of blood in the tube.

4. Centrifuge at 4°C, 2000 rpm for 15 min. Breaks off!! 5. While the tubes are centrifuged put the droplet of blood that you saved

on a slide. Look at the rosettes in microscope. Take a photo. 6. After centrifugation the T-lymphocytes are in the pellet while the ”non-

T-fraction” is in the interphase. Carefully suck up the interphase and put it in a 15 ml-tube. Dilute the non-T-fraction to 15 ml with R5 and wash (10 min, 1000 rpm). Resuspend in 2 ml and count the cells (dilute 100 times for the counting). After that, resuspend the cells to a density of 10*106 cells/ml. Take out samples of 0,5 x 106 cells (=50ul) and add to 3 tubes (”FACS tubes”). Mark the tubes!! (D1, D2 resp. D3)

7. Take away the rest of the supernatant (from step 6.). The pellet (T

lymphocytes) could be quit loose, so be careful! Prepare a pipette for 500 µl with a new tip. Add 4 ml dH2O/tube, pipette once up and down and then add 500µl 10x NaCl (1,4 M) within 10 seconds!

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8. Add R5 up to 15 ml in each tube and centrifuge 1250 rpm for 10 min. Pool the cell suspensions from the different tubes to a 50 ml-tube.

9. Wash the cells one more time in 10 ml R5. Resuspend the cells in 2 ml.

Count the cells (diluted 100 times for the counting). Resuspend the cells to 10 x 106/ml in R5. Count carefully! Save half a million of cells (50 µl) in 6 different 5 ml tubes (”FACS tubes”). Mark the tubes!! (C1, C2, C3, C4, resp. C5)

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DAY 2: Binding of adherent cells to plastic surface 1. Take out the monocytes from the incubator. Check that the monocytes

have come off from the plastic surface. If not, try to hit the flask gently. Transfer the cell suspension to a 50 ml tube and centrifuge. The pellet will be really small so be careful when you aspirate the suspension on the top. Resuspend the cells in 0,5ml and count (dilute 10 times). Dilute to a concentration of 10*106 cells/ml. Transfer 0,5 x 106 cells to three different 5 ml tubes (50 µl/tube) and mark the tubes with B1, B2 resp B3.

Surface staining of fractionated PBMC 1. Add 1 µl mouse Ig blocking (100ul/ml) solution to each of the 14

samples. 2. The staining of the cell is performed in a total of 100 µl where 50 µl is

cell suspension and 50 µl is an antibody cocktail. Add 50 µl of each prepared antibody cocktail with marked antibodies to the cell suspension as follow:

Sample: Cocktail: A1, B1, C1, D1 unstained A2, B2, C2, D2 isotype-control A3, B3, C3, D3 CD3/CD19/CD14 C4 CD3/CD4/CD8 C5 CD4/ CD8/CD45RO 3. Incubate on ice for 30 min. 4. Wash 1 x 3 ml with PBS/1% BSA (5 min, 1250 rpm). 5. Resuspend the cells in 500 µl PBS/1%BSA. 6. Run FACS-analysis.

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Staining with fluorescent labeled antibodies for flow cytometry analysis By using fluorescent labeled antibodies one can decide what type of cells and their internal relations in a sample. By sampling at different stages in the purification process, one can follow the purification. Each fraction (A, B, C, D) was stained with the following antibodies:

Sample 1 (A, B, C, D): Unstained cells

Sample 2 (A, B, C, D): mouse IgG1 FITC mouse IgG1 PE

(isotype control)

Sample 3 (A, B, C, D): α CD3-APC α CD19-FITC α CD14-PE

Sample C4: α CD3 APC α CD4 PE

α CD8 FITC

Sample C5: α CD4 PE α CD8 FITC

α CD45RO APC

α CD19 stain B-cells α CD14 stain monocytes α CD3 stain T-cells (both CD4- and CD8-positive T-cells) α CD4 stain T-helper cells of type TH1 and TH2 α CD8 stain cytotoxic T-lymphocytes (CTLs) α CD45RO stain memory T-cells

Which cell type is the dominating one in each fraction? A. …………………………….. B. …………………………….. C. …………………………….. D. ……………………………..