01. Direct immunofluorescence staining of cells and blood for Flow Cytometry
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 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:
Substrate | For Use With | Product Codes | Stop Solution |
|---|---|---|---|
TMB | HRP-Conjugated Antibodies | 2M H2SO4 | |
OPD | HRP-Conjugated Antibodies | 1M H2SO4 | |
pNPP | Alkaline Phosphatase-Conjugated Antibodies | 1M NaOH |
Method
- Prepare cells appropriately. Adjust the cell suspension to a concentration of 1 x 106 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]. - Aliquot 100 μl of the cell suspension [whole blood] into as many test tubes as required.
- Add antibody at the recommended dilution (see specific datasheets). Mix well and incubate at room temperature for 30 minutes.
- 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 BUF04 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.] - 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.
Please contact AbD Serotec’s Technical Services Department for details of available reagents.
