sample preparation

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Flow Cytometry Sample Preparation

In a typical flow cytometry sample preparation, phosphate buffered saline (PBS) is a common suspension buffer and the most straightforward samples for flow cytometry include non-adherent cells from culture, bacteria, yeast, blood and tissues.

For cell culture, the growth of cells should better be 105–107 cells/ml to prevent the flow cytometer from clogging up for the sorting speed is about 2,000–20,000 cells/second.

For the blood, red cells are usually removed by a simple lysis step; then lymphocytes, granulocytes and monocytes are quickly identified by their FSC/SSC characteristics.

For solid tissues e.g. liver or tumors, in order to produce single cells, the solid material must be disaggregated either mechanically or enzymatically.

Mechanical disaggregation involves passing a suspension of chopped tissue through a fine-gauge needle several times, followed by grinding and sonication as necessary.

Enzymes are used to disrupt protein-protein interactions and the extracellular matrix that hold cells together. Their action is dependent on factors including pH, temperature and co-factors, so take care when choosing an enzyme.

To study intracellular components e.g. cytokines by flow cytometry, the plasma membrane of the cell must be permeabilized to allow dyes or antibody molecules through while retaining the cell’s overall integrity. Low concentrations (up to 0.1%) of non-ionic detergents like saponin are suitable. In summary, the method for sample preparation will depend on the starting material and the nature of the epitope.

1. Preparation of cells

5. Whole blood protocol for analysis of intracellular cytokines

2. Direct immunofluorescence staining of cells and blood

6. Direct staining of intracellular antigens

3. Indirect immunofluorescence staining of cells and blood

7. Direct staining of intracellular antigens: methanol method

4. Staining lambda and kappa chains in whole blood

 

 

1. Preparation of cells

(a) Cells stored in liquid nitrogen

1 Prepare PBS/BSA buffer (phosphate buffered saline pH 7.4 and 1% BSA).

2 Carefully remove cells from liquid nitrogen storage.

3 Thaw rapidly using PBS/BSA buffer and place into a 15 ml conical centrifuge tube.

4 Centrifuge at 400 g for 5 minutes.

5 Discard supernatant and resuspend pellet in an appropriate amount of PBS/BSA buffer.

(b) Tissue culture cell lines in suspension

1 Prepare PBS/BSA buffer (phosphate buffered saline pH 7.4 and 1% BSA).

2 Decant cells from tissue culture flask into 15 ml conical centrifuge tube(s).

3 Centrifuge at 400 g for 5 minutes.

4 Discard supernatant and resuspend pellet in 10 ml of PBS/BSA.

5 Centrifuge at 400 g for 5 minutes.

6 Discard supernatant and resuspend pellet in an appropriate amount of PBS/BSA.

(c) Adherent tissue culture cell lines

1 Prepare PBS/BSA buffer (phosphate buffered saline pH 7.4 and 1% BSA).

2 Harvest cells by gentle scraping using 2 ml of PBS/BSA buffer.

3 Transfer cells to a 15 ml conical tube and add buffer up to 10 ml.

4 Centrifuge at 400 g for 5 minutes.

5 Discard supernatant and resuspend pellet in fresh PBS/BSA (10 ml).

6 Centrifuge at 400 g for 5 minutes.

7 Discard supernatant and resuspend pellet in an appropriate amount of PBS/BSA buffer.

(d) Preparing cells from solid/lymphoid tissues

1 Place tissue on a sterile Petri dish. Remove cells by gently perfusing the tissue using a syringe and needle containing approximately 15 ml of PBS/BSA (phosphate buffered saline pH 7.4 and 1% BSA).

2 Transfer the cell suspension from the Petri dish into a 15 ml conical centrifuge tube.

3 Centrifuge at 400 g for 5 minutes.

4 Discard the supernatant and resuspend the pellet in PBS/BSA.

5 Add 10 ml of ammonium chloride lysis buffer.

6 Mix and incubate for 2 minutes. DO NOT EXCEED THIS TIME.

7 Centrifuge at 400 g for 5 minutes.

8 Add 10 ml of PBS/BSA and mix.

9 Centrifuge again at 400 g for 5 minutes.

10 Discard the supernatant and resuspend the pellet to a final volume of 10 ml with PBS/BSA.

11 Count cells using a hemocytometer.

12 Adjust the cell suspension, if necessary, to give a final count of 0.7–1.2×107 cells/ml.

 

2. Direct immunofluorescence staining of cells and blood

This technique is applicable where the fluorochrome is directly linked to the primary antibody e.g. PE, FITC and Alexa Fluor® conjugates.

Note. Specific methodology for blood appears in [ ] brackets.

1 Prepare cells appropriately (see section 1). Adjust the cell suspension to a concentration of 1 × 106 cells/ml with PBS/BSA buffer (phosphate buffered saline pH 7.4 and 1% BSA).

[Whole blood samples may be used undiluted unless the cell count is high e.g. as in leukemia. EDTA and heparin are preferred anti-coagulants].

2 Aliquot 100 μl of cell suspension [whole blood] into as many test tubes as required.

3 Add antibody at the recommended dilution (see specific datasheets). Mix well and incubate at room temperature for 30 minutes.

4 Wash cells with 2 ml of PBS/BSA, centrifuge at 400 g for 5 minutes and discard the resulting supernatant.

[To the blood suspension add freshly prepared red cell lysis buffer e.g. 2 ml of AbD Serotec’s Erythrolyse and mix well. Incubate for 10 minutes at room temperature. Centrifuge at 400 g for 5 minutes and discard the supernatant].

Resuspend cells in 0.2 ml of PBS/BSA or with 0.2 ml of 0.5% paraformaldehyde in PBS/BSA if required.

Acquire data by flow cytometry. Appropriate standards should always be included e.g. an isotype-matched control sample.

3. Indirect immunofluorescence staining of cells and blood

This technique is applicable where using unconjugated or biotin-conjugated monoclonal and polyclonal antibodies. A secondary reagent must be used to visualize the primary antibody e.g. avidin in the case of biotin.

Note. Specific methodology for blood appears in [ ] brackets.

1 Prepare cells appropriately (see section 1). Adjust the cell suspension to a concentration of 1 × 106 cells/ml with PBS/BSA buffer (phosphate buffered saline pH 7.4 and 1% BSA).

[Whole blood samples may be used undiluted unless the cell count is high e.g. as in leukemia. EDTA and heparin are preferred anti-coagulants].

2 Aliquot 100 μl of cell suspension [whole blood] into as many test tubes as required.

3 Add primary antibody at the recommended dilution (see specific datasheets). Mix well and incubate at room temperature for 30 minutes.

4 Add 2 ml of PBS/BSA buffer, centrifuge at 400 g for 5 minutes and discard the resulting supernatant.

5 Add an appropriate secondary reagent at the recommended dilution (see specific datasheets). Mix well and incubate at room temperature for 30 minutes.

6 Wash cells with 2 ml of PBS/BSA, centrifuge at 400 g for 5 minutes and discard the supernatant.

[To the blood suspension add freshly prepared red cell lysis buffer e.g. 2 ml of AbD Serotec’s Erythrolyse and mix well. Incubate for 10 minutes at room temperature. Centrifuge at 400 g for 5 minutes and discard the supernatant].

7 Resuspend cells in 0.2 ml of PBS/BSA or with 0.2 ml of 0.5% paraformaldehyde in PBS/BSA if required.

8 Acquire data by flow cytometry. Appropriate standards should always be included e.g. an isotype-matched control sample.

4. Staining lambda and kappa chains in whole blood

This method should be used with directly-conjugated dual-color reagents recognizing human kappa and lambda immunoglobulin light chains. Detection of immunoglobulin expression specifically on B lymphocytes requires a procedure to remove blood serum immunoglobulins that would otherwise cause interference.

1 Collect blood in an anti-coagulant e.g. EDTA, heparin or acid-citrate dextrose.

2 Aliquot 2–3 ml of whole blood into a 25 ml universal container. Then add 20–25 ml of PBS/BSA (phosphate buffered saline pH 7.4 and 1% BSA), pre-warmed to 37°C, and mix well.

3 Centrifuge at 400 g for 5 minutes. Carefully aspirate the supernatant taking care not to disturb the cell pellet. Resuspend the pellet in the residual supernatant.

4 Repeat the wash (steps 2 and 3) twice.

5 Aliquot 100 μl of washed blood into the required number of test tubes. Add antibody at the recommended dilution (see specific datasheet). Mix well and incubate at room temperature for 30 minutes.

6 Add a red cell lysis buffer e.g. 2 ml of AbD Serotec’s Erythrolyse and mix well. Incubate for 10 minutes at room temperature. Centrifuge at 400 g for 5 minutes and discard the supernatant.

7 Wash cells with 2 ml of PBS/BSA, centrifuge at 400 g for 5 minutes and discard the supernatant.

8 Resuspend cells in 0.2 ml of PBS/BSA or with 0.2 ml of 0.5% paraformaldehyde in PBS if required.

9 Acquire data by flow cytometry. Appropriate standards should always be included e.g. an isotype-matched control sample.

5. Whole blood protocol for analysis of intracellular cytokines

This is a rapid and simple approach to the analysis of intracellular cytokines by flow cytometry. It permits the analysis of small samples, and avoids any possibility of generating artefactual results during the separation of peripheral blood cells by density gradient centrifugation. The stimulation conditions described are suitable for IFN gamma, IL-2 and TNF alpha. Different conditions may be needed for other cytokines.

The procedure requires a reagent kit to fix and permeabilize cells. There are several available but we recommend LeucopermTM.

Note. All blood samples must be collected into heparin anti-coagulant. EDTA interferes with the cell stimulation process and, therefore, must be avoided.

1 Aliquot 0.5 ml of blood separately into 2 tubes, then add 0.5 ml of cell culture medium (without any additives) to each sample.

2 To one tube (the resting population), add monensin to a final concentration of 3 mM.

3 To the other tube (activated cells), add PMA, ionomycin and monensin to a final concentration of 10 ng/ml, 2 mM and 3 mM, respectively.

4 Incubate for 2–4 hours at 37°C in a 5% CO2 atmosphere.

5 At the end of the incubation period aliquot 100 μl samples into the appropriate number of tubes.

6 Add cell surface antibodies at this stage (if needed for your experiment) and incubate for 15 minutes.

7 Add 100 μl of LeucopermTM Reagent A per tube and incubate for 15 minutes. This reagent fixes cells in suspension.

8 Wash twice with PBS containing 0.1% sodium azide and 1% BSA.

9 Add 100 μl of LeucopermTM Reagent B (permeabilizes cells) and the required anti-cytokine antibodies.

10 Incubate for 20 minutes.

11 Wash twice using the PBS buffer, and analyze by flow cytometry.

6. Direct staining of intracellular antigens

The detection of intracellular antigens requires a cell permeabilization step prior to staining. The method described below produces excellent results in our hands; however other permeabilization techniques have been published, and may also be successfully used for this application.

1 Harvest cells and determine total number present.

2 Wash twice in wash buffer (PBS containing 1% BSA and 0.1% sodium azide).

3 If required, perform staining of cell surface antigens using appropriate directly conjugated monoclonal antibodies at this stage. Following staining, wash cells once in PBS and discard the supernatant.

4 Resuspend cells in LeucopermTM Reagent A (cell fixation agent) using 100 μl per 1 × 106 cells. Incubate for 15 minutes at room temperature.

5 Wash once in wash buffer.

6 Resuspend cells in LeucopermTM Reagent B (cell permeabilization agent) using 50 μl per 1 × 106 cells.

7 Aliquot 50 μl of cell suspension into the required number of tubes containing directly-conjugated antibodies. Incubate for 30 minutes at room temperature.

8 Wash once in wash buffer, and then resuspend in 0.25 ml of 0.5% paraformaldehyde in PBS.

9 Store at 4°C until acquisition on the flow cytometer, preferably within 24 hours.

7. Direct staining of intracellular antigens: methanol method

Methanol modification is particularly suitable for the detection of some nuclear antigens, such as PCNA and Ki-67.

Note. Phycoerythrin conjugates are not suitable for the detection of cell surface antigens using this method.

1 Harvest cells and determine the total number present.

2 Wash twice in wash buffer (PBS containing 1% BSA and 0.1% sodium azide).

3. If required, perform staining of cell surface antigens using appropriate directly conjugated monoclonal antibodies at this stage. Following staining, wash cells once in PBS and discard the supernatant.

4 Resuspend cells in cold (2–8°C) LeucopermTM Reagent A using 100 μl per 1 × 106 cells. Incubate for 10 minutes at 2–8°C.

5 Add 500 μl of cold absolute methanol, vortex and incubate for 10 minutes at 2–8°C.

6. Wash once in wash buffer.

7 Resuspend cells in LeucopermTM Reagent B using 100 μl per 1 × 106 cells.

8 Aliquot 50 μl of cell suspension into the required number of tubes containing directly conjugated antibodies. Incubate for 30 minutes at room temperature.

9 Wash once in wash buffer, and resuspend in 0.25 ml of 0.5% paraformaldehyde in PBS.

10 Store at 4°C until acquisition on the flow cytometer, preferably within 24 hours.