Diatec - Flow Methods

Diatec AS Gaustadalléen 21 N-0349 Oslo, Norway	Tel: (+47) 22 95 86 25 Fax: (+47) 22 95 86 49 diatec@diatec.com
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Flow cytometry methods page

This page is intended to give some hints and tips on methods and sample preparation for Flow Cytometry. As all labs have their own protocols and methods, it is always important to be able to test out a new antibody before using it. For this purpose, we may supply you with a free 10 test sample of the desired antibodies.
Contact us: diatec@diatec.com

Sample preparation
- Staining of whole blood
- Staining of cells
Running flow cytometry
Compensation
Presentation of data

Sample preparation

Staining of whole blood

General recommendation: Whole blood, no wash methods recommended by some suppliers are easy, and may at first glance look time saving and convenient for routine analysis. However, it is important to be aware of that the presence of unreacted antibodies may cause higher background, especially with low count epitopes. This makes the analysis conditions less optimal, giving a lower signal/background ratio than methods with wash. An other aspect is that with a higher volume the analysis time will be longer, thus the time consuming argument will be only partially true. Spinning down, removing unreacted antibodies and resuspending in a smaller volume of buffer will for large sample numbers be as quick as analysing unwashed, large volume samples.
Therefore, we always recommend one wash after the antibody incubation step. ¹
This recommendation is valid for antibodies from all suppliers.

Whole blood staining with FITC, PE or APC-conjugates:
100 m L blood + 20 m L mAb
Follow instruction on whole blood lysing buffer. Wash once with 2 mL wash buffer to yield optimal results.

Staining of cells

Wash buffer PBS-BR: PBS containing
1% BSA (Sigma)
1% rabbit serum (Gibco)
This wash buffer prevents non-specific binding of mAbs to sample.

Staining cell preparations with FITC, PE or APC-conjugates:
50 m L (500 000) cells + 20 m L mAb

Wash cells once with PBS-BR (Spin down at 400g for 5 min).
Resuspend cell pellet in PBS-BR to a concentration of 10 mill/mL. Incubate for 15 min. on ice to block unspecific sites.
Spin down at 400g for 5 min., resuspend cell pellet in PBS-BR to a concentration of 10 mill/mL.
Use 0.5-1 mill. cells (50-100µL) per test.
Add 20µL of each antibody per 50無 (0.5 mill.) cells, incubate for 30 min. dark on ice.
Wash twice with 2 mL PBS-BR, discard supernatant
Resuspend in 200-300µL of the sheath fluid for the flow cytometer. Run samples on flow cytometry same day.
optional, if samples need to be stored, fix cells in 200-300µL of a 1% paraformaldehyde in PBS solution. Time from paraformaldehyde fixation to analysis should be minimum 4 hours to avoid signal drift, as cell scatter changes slightly during fixation.

Running flow cytometry

General guidelines for running flow cytometry. For quality control of apparatus, we recommend that the alignment of the flow cytometer is controlled and adjusted using calibration beads for the actual instrument at the beginning of each day. This is done to verify that day-to-day variations are as low as possible.
For daily check of cells and protocols, use

negative control: unstained cells. The unstained sample shold lie within the first quadrant of the diagram, and should not be stacked up against the axis.

isotype control: cells incubated with irrelevant mAb of correct isotype and concentration. Set a gate (/quadrant) relative to these, it is usual to leave about 2% of cells in the gate.

positive control: cells stained with mAbs against a known positive epitope to verify signal output.

Protocols

Graph A: May be forward scatter (FS) against side scatter (90 degree scatter, 90LS). This gives a plot describing size (FS) vs. granularity (90LS), doing it possible to gate on specific cell types.
OR the plot may be 90LS versus a signal specific for the desired cell type, e.g. CD45.
Set a gate on the interesting population (e.g. lymphocytes).
Graph B-C-etc: For each color analysed, one might set up an intensity vs. count- and an intensity vs. side scatter plot. Usually, log intensity is used. Make combined plots of e.g. PMT2 (FITC) vs. PMT3 (PE) etc. for the parameters to be analysed. These plots might be gated on the interesting population (e.g. lymphocytes) from Graph A.
Adjust voltage so that the negative (unstained) cells are in the first quadrant, read: on screen. Do not pack the negative population up against the edge of the graph.
Compensation of two-parametric runs is important because a fluorescent marker, e.g. the FITC, also have some emmission in other channels, e.g. the PE channel, and this has to be subtracted to avoid creating a false result. Compensation must be done running single-stained samples in two-parametric plots. For good instructions, see the compensation site of Mario Roederer, Stanford University

Run samples. Record percent positive and mean peak channel or peak position in one parametric plots, or record percentage of cells in quadrants for multi-parameter runs.

Presentation of data

Data from flow cytometry runs are saved on "list mode" form (.lmd). Data are analysed directly through the flow software. To do analysis of your files later, you may use WinMDI which reads the .lmd-format. WinMDI may be used for creating graphs, comparisons etc. This is freeware, available to download from The Scripps research institute (TSRI)

References and selected links:

Current protocols in cytometry, Managing Editor: J. Paul Robinson / Zbigniew Darzynkiewicz (ed) / Phillip Dean (ed) / Alberto Orfao (ed) / Peter Rabinovitch (ed) / Carleton Stewart (ed) / Hans Tanke (ed) / Leon Wheeless (ed), (Series Editior: J. Paul Robinson)John Wiley & Sons Inc., 1997, ISBN 0-471-16132-2
Practical Flow Cytometry, Howard Shapiro, John Wiley and Sons, 1994, ISBN 0-471-30376-3
Consenseus document on leukemia phenotyping, European Working Group on Clinical Cell Analysis
Recommended guidelines standards for leukemia phenotyping, Australasian Flow Cytometry group