This protocol describes intracellular SAP and XIAP stain in different lymphocyte populations by 5 colour flow cytometry.

80μl whole blood (EDTA) or 1x10⁶ peripheral blood mononuclear cells (PBMC) obtained by density gradient centrifugation of EDTA blood.

EDTA-wash buffer (PBS w/o Ca²⁺ and Mg²⁺, 2% FCS, 2 mM EDTA)

IntraPrep (Beckman Coulter Nr. A07803)

centrifuges

minimum 5 colour flow cytometer

1,5 hours

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Step 1: Label sample tubes: control isotype, control SAP/XIAP, patient (initials or other identifier like lab number) isotype, and patient SAP/XIAP and add 80ul of blood (or 2x10⁵ PBMC) to each tube.

Step 2: Add 100 μl of IntraPrep reagent 1 to all tubes. Vortex vigorously immediately after each addition. Incubate for 15 minutes at room temperature (18 – 25°C).

Step 3: Wash one time with wash buffer (300x g, 6 min) at room temperature and remove the supernatant by aspiration.

Step 4: Add 100 μl of IntraPrep reagent 2 to all tubes. DO NOT VORTEX, leave reagent 2 to diffuse naturally into the cell pellet. Incubate for 5 minutes at room temperature (18 – 25°C) WITHOUT SHAKING. Shake slowly by hand for 2 to 3 seconds.

Step 5: Add the following antibodies to the indicated tubes and incubate for 30 min at 4°C:

Isotype SAP/XIAP

2 μl mouse IgG2a Isotype 2 μl anti-SAP

5 μl mouse IgG1 Isotpye 5 μl anti-XIAP

Step 6: Wash two times with wash buffer (300x g, 6 min) at room temperature. Remove the supernatant by aspiration.

Step 7: Add the following antibodies to all tubes and incubate for 15 min at 4°C:

4 μl rat-anti-mouse IgG2a FITC (binds to anti-SAP and its isotype)

2 μl goat-anti-mouse IgG1 PE (binds to anti-XIAP and its isotype)

Step 8: Wash two times with wash buffer (300x g, 6 min) at room temperature and remove the supernatant by aspiration.

Step 9: Add the following surface antibodies to all tubes and incubate for 20 min at 4°C:

4 μl anti-CD3 PerCP

0,5 μl anti-CD56 APC

2 μl anti-CD19 PE-Cy7

Step 10: Wash one time with wash buffer (300x g, 6 min) at room temperature and resuspend cell pellet in about 300µl PBS for analysis by the flow cytometer.

Cells from region R1, defining the lymphocyte population, are displayed in a plot CD3 PerCP vs. CD56 APC and a plot CD3 PerCP vs. CD19 PE-Cy7, both containing quadrant regions that define CD56⁺CD3^- NK (Q1), CD3⁺CD56^- T (Q4) and CD3^-CD19⁺ B (Q5) cells.

Combined regions R1 and Q1 define the “NK-cell” gate, combined regions R1 and Q4 define the “T-cell” gate and combined regions R1 and Q5 define the “B-cell” gate. Histogram plots display SAP FITC and XIAP PE expression of each lymphocyte population.

Suggested Acquisition setting: Events saved: 50000 within the lymphocyte population; time limit of 5 min.. It is recommended to record all cells, or at least all lymphocytes, in order to re-examine the specificity of unexpected results.

Cells are gated as described under Data acquisition. Adjust regions to the populations if necessary.

Define isotype stained NK cells of the healthy control as "SAP negative" using a histogram plot of SAP FITC expression. To compare isotype with SAP expression do the same for the SAP/XIAP tube (histogram overlay is recommended). Do the same for T and B cells. B cells do not express SAP.

Analyse XIAP expression accordingly.

SAP and XIAP expression are interpreted by visual comparison of the histograms to the respective lymphocyte population of the day control. There are no defined minimum levels of SAP/XIAP expression. B cells don't express SAP. Heterozygote female carriers of SAP or XIAP mutation can show double peaks due to the presence of a SAP/XIAP positive and negative lymphocyte population. In several cases the interpretation is difficult, as SAP or XIAP expression can be only reduced or even normal in deficient patients. The interpretation of the results requires awareness of the medical history of the patient. If there is a high clinical suspicion, the genetic analysis is recommended even in case of a normal flowcytometric staining.

Beside standard methods of quality control, a healthy control should be run with this test as positive control to detect staining errors. It is recommended that the control sample is collected at the same time as the patient sample and they are transported together to control for delays and temperature changes in transport.

At least 3000 events within the different lymphocyte population gates are necessary for a clear interpretation.

In cases of high unspecific signal in the isotype controls the assay should not be interpreted, as staining errors are possible.

High non-specific signal in the isotype controls can occur especially in whole blood samples, the test should be repeated with isolated PBMC.

Wash steps should be performed thoroughly as free antibodies have to be removed completely to avoid wrong labelling.

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