Flow Cytometry

Note: Phycoerythrin conjugates are not suitable for the detection of cell surface antigens using this method due to damage of the RPE at low temperatures.

The detection of intracellular antigens requires a cell permeabilization step prior to staining. The method described below has been found to provide excellent results in our hands; however other permeabilization techniques have been published, and may also be successfully used in this application. This modification is particularly suitable for the detection of some nuclear antigens, such as PCNA and Ki67. In some cases specific recommendations are provided on product datasheets, and this method should always be used in conjunction with product and batch specific information provided with each vial. Please note that a certain level of technical skill and immunological knowledge is required for the successful design and implementation of these techniques - these are guidelines only and may need to be adjusted for particular applications.

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

Reagents ‎

Leucoperm (BUF09) ‎ Wash Buffer, Phosphate Buffered Saline (PBS) containing 1% PBS and 0.09% Sodium azide. ‎

1. Harvest cells and determine the total number present. Adjust cell suspension to a concentration of 1 x 107 cells/ml in PBS containing 1% BSA.
   ‎[Whole blood samples may also be used. AbD Serotec recommends the use of EDTA anti-coagulant in ‎these circumstances, although satisfactory results may be obtained using heparin or acid-citrate ‎dextrose.] ‎
2. Add 100 μl of cell suspension [whole blood] to the appropriate number of test tubes. ‎
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/BSA and discard the supernatant.
4. Resuspend cells in cold (2-8°C) LeucopermTM Reagent A using 100 μl per 1 x 106 cells. Incubate for 10 minutes at 2-8°C.
5. Add 500 μl of ice cold absolute methanol, vortex and incubate for 10 minutes at 2-8°C.
6. Add 3ml PBS and centrifuge for 5 minutes at 300 g.‎
7. Remove supernatant and add 100 μl LeucopermTM Reagent B (cell permeabilization agent) ‎per 1 x 106 cells and add 10 μl of the appropriate, directly conjugated antibody. ‎
8. Wash once in wash buffer, and then resuspend in sheath fluid for immediate analysis or with 0.25 ml of 0.5% formaldehyde in PBS/BSA if required.
   [To the blood suspension add freshly prepared red cell lysis buffer e.g. 2 ml of AbD Serotec’s Erythrolyse BUF04 and mix well. Incubate for 10 minutes at room temperature. Centrifuge at 400 g for 5 minutes and discard the supernatant.]
9. Acquire data by flow cytometry. Analyse fixed cells ‎within 24 hours.

Applicable where the fluorochrome is directly linked to the primary antibody e.g. RPE, FITC and Alexa Fluor® conjugates. Please note that RPE conjugates should be handled in the dark throughout.

This technique is applicable when using unconjugated or biotin-conjugated monoclonal and polyclonal antibodies recognizing cell surface antigens. A conjugated secondary reagent must be used to visualize the primary antibody, e.g. streptavidin in the case of biotin. Please note that RPE conjugates should be handled in the dark throughout.

This method provides a general procedure for use with the majority of AbD Serotec reagents. In some cases specific recommendations are provided on product datasheets, and these methods should always be used in conjunction with the product and batch specific information provided with each vial. Please note that a certain level of technical skill and immunological knowledge is required for the successful design and implementation of these techniques - these are guidelines only and may need to be adjusted for particular applications.

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

Reagents:

PBS/BSA Phosphate Buffered Saline pH 7.4 with 1% Bovine Serum Albumin

Method

1. Prepare cells appropriately. Adjust the cell suspension to a concentration of 1 x 107 cells/ml with PBS/BSA buffer.
   [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 the 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 BUF04
7. and mix well. Incubate for 10 minutes at room temperature. Centrifuge at 400 g for 5 minutes and discard the supernatant. Wash 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/BSA if required.
9. Acquire data by flow cytometry. Analyze fixed cells within 24 hours.

Appropriate standards should always be included, e.g. an isotype-matched control sample. It may also be useful to include a control in which no primary antibody is used at all, to determine any non-specific binding of the secondary reagent to the target cells.

For use with AbD Serotec’s directly-conjugated dual colour reagents recognising human kappa and lambda immunoglobulin light chains.

The immunofluorescent staining of immunoglobulin expression by B lymphocytes in whole blood requires a procedure to remove serum immunoglobulins (which otherwise interfere and block staining with immunoglobulin-specific antibodies).

Note: Specific methodology for blood appears in [] brackets

Reagents:

PBS/BSA

Phosphate Buffered Saline pH 7.4 20mM containing glucose and 1% Bovine Serum Albumin

This method provides a general procedure for use with the majority of AbD Serotec reagents. In some cases specific recommendations are provided on product datasheets, and these methods should always be used in conjunction with product and batch specific information provided with each vial. Please note that a certain level of technical skill and immunological knowledge is required for the successful design and implementation of these techniques - these are guidelines only and may need to be adjusted for particular applications.

1. Collect blood into appropriate anti-coagulant - AbD Serotec recommends the use of EDTA, although satisfactory results may also be obtained using 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, 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, and resuspend the pellet in the residual supernatant.
4. Repeat steps 3 and 4 twice more (three washes in total).
5. Aliquot 100 ul of the washed blood into the required number of test-tubes. Add appropriate volume of AbD Serotec antibody at the recommended dilution (see specific datasheet). Mix well and incubate at room temperature for 30 minutes.
6. Add 2 ml of freshly prepared AbD Serotec Erythrolyse (BUF04 and mix well. Incubate for 10 minutes at room temperature. Centrifuge at 400 g for 5 minutes. Discard supernatant.
7. Wash 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/BSA if required.
9. Acquire data by Flow Cytometry.

For the detection of cell cycle antigens such as Ki-67, PCNA and BrdU, methanol modification is recommended – see protocol F5

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 used successfully for this application.

In some ‎cases specific recommendations are provided on product datasheets, and these methods should always ‎be used in conjunction with product and batch specific information provided with each vial. Please note ‎that a certain level of technical skill and immunological knowledge is required for the successful design ‎and implementation of these techniques - these are guidelines only and may need to be adjusted for ‎particular applications. ‎

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

Reagents

1. Leucoperm (BUF09)

2. Wash Buffer
Phosphate Buffered Saline (PBS) containing 1% PBS and 0.09% Sodium azide.

1. Harvest cells and determine the total number present. Adjust cell suspension to a concentration of 1 x 107 cells/ml in PBS containing 1% BSA.
   [Whole blood samples may also be used. AbD Serotec recommends the use of EDTA anti-coagulant in these circumstances, although satisfactory results may be obtained using heparin or acid-citrate dextrose.]
2. Add 100 μl of cell suspension/whole blood to the appropriate number of test tubes.
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/BSA and discard the supernatant.
4. Resuspend cells in LeucopermTM Reagent A (cell fixation agent) using 100 μl per 1 x 106 cells. Incubate for 15 minutes at room temperature.
5. Add 3ml PBS and centrifuge for 5 minutes at 300 g.
6. Remove supernatant and add 100 μl LeucopermTM Reagent B (cell permeabilization agent) per 1 x 106 cells and add 10 μl of the appropriate, directly conjugated antibody.
7. Vortex at low speed for 1-2 seconds and incubate for 30 minutes at room temperature.
8. Wash once in wash buffer, and then resuspend in sheath fluid for immediate analysis or with 0.25 ml of 0.5% formaldehyde in PBS/BSA if required.
   [To the blood suspension add freshly prepared red cell lysis buffer e.g. 2 ml of AbD Serotec’s Erythrolyse BUF04 and mix well. Incubate for 10 minutes at room temperature. Centrifuge at 400 g for 5 minutes and discard the supernatant]
9. Acquire data by flow cytometry. Analyse fixed cells within 24 hours.