Administración de Riesgos, (continuación)

2.14.1 Human CD8 + T- cell Magnetic bead panel (Milliplex® map Kit,

EMD)

Milliplex® map is based on the Luminex xMAP® technology and in the present study, a 17-plex panel was used (EMD millipore’s Milliplex® map Human CD8 + T-cell magnetic bead panel). This panel was used for the simultaneous quantification of the following in the culture supernatant samples: Perforin and Granzyme B.

2.14.1.1. Preparation of tissue culture supernatant and reagents for immunoassay

Two hundred µL of each sample was stored in polypropylene cryovials (Merck, South Africa) at -20°C in the BSL3 facility after each stimulation. Before running the assay all the samples were filtered using 0.2µm Millex® syringe filters (EMD Millipore, USA). The samples were thawed at RT and then vortexed (Vortex whirl mixer, Stuart® equipment, UK) for 5 seconds. Thereafter the samples were centrifuged (Eppendorf, Germany) for 2 minutes at 2000 rpm.

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2.14.1.2 Preparation of reagents for immunoassay

Antibody-immobilized beads

Each antibody-bead vial was sonicated (Bransonic Ultrasonic bath sonicator) (Sigma-Aldrich, South Africa) for 30 seconds and then vortexed (Stuart® equipment, UK) for 1 minute. Thereafter 80µL of each antibody-bead vial was added to a mixing bottle and bead diluent was added to make up the final volume to 5.6mL (since two plates will be run). The mixed beads were vortexed (Stuart® equipment, UK) well before use.

Quality controls (QC)

Quality control 1 and 2 was reconstituted with 250µL deionized water. The vial was inverted several times and vortexed (Stuart® equipment, UK). The vials were left for 5-10 minutes before use.

Wash buffer

The 10x wash buffer was brought to RT and mixed to bring all the salts into solution and then 60mL of the wash buffer was diluted with 540mL deionized water.

Preparation of standards

Six polypropylene tubes were labelled, Standard 1 to Standard 6. One hundred and fifty µL of Assay buffer was added to all of the tubes. Serial dilutions were then prepared by adding 50µL of the reconstituted Standard 7 to Standard 6 (mix well) and then 50µL of Standard 6 to Standard 5 etc. as indicated in figure 2.10.

Table 2.6: Preparation of CD8 + T cell standard (standard 7) and working standards.

Standard tube Volume of Deionized Water

to add

Volume of Standard to add

Standard 7 250µL 0

Standard tube Volume of Assay Buffer to

add

Volume of Standard to add

Standard 6 150µL 50µL of Standard 7 Standard 5 150µL 50µL of Standard 6 Standard 4 150µL 50µL of Standard 5 Standard 3 150µL 50µL of Standard 4 Standard 2 150µL 50µL of Standard 3 Standard 1 150µL 50µL of Standard 2

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Figure 2.10: The workflow for the preparation of the working standards. Adapted from booklet Human

CD8+ T-cell magnetic Bead panel kit booklet (EMD Millipore, USA).

2.14.1.3 Assay procedure for Human CD8 + T- Cell Magnetic Bead panel

All reagents were allowed to reach RT (20-25°C) before use. The assay was performed as indicated in figure 2.11. After the 16-18 hour incubation step, the plates were washed twice using Bio-plex Pro™ wash station (Bio-rad, USA).

Page | 45 The plates were analysed on the Luminex® Bioplex 200 system (Bio-rad, USA) with the program, Bioplex manager. Calibration and verification was done as part of the daily setup. The plate layout is indicated in figures 2.12 and 2.13.

Figure 2.12: The 96 well -plate layout of plate 1 (A) and plate 2 (B) for Human CD8 + T cell magnetic bead

assay. Standard 0 = blank, standard 1 = low concentration standard, standard 6 = Highest concentration std, QC1 = Assay Quality control 1, QC2 = Assay Quality Control 2. Evaluate QC1 and QC2 as recommended by kit manufacturer and the interpolate control just like any other sample. Each circle represents a well. Each sample was analysed in triplicate for each time point as well as for stimulations with and without hIL-2.

A

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2.14.2 Human Cytokine/Chemokine magnetic bead panel (Milliplex® map

kit, EMD)

This panel was used for the simultaneous quantification of the following cytokines in the culture supernatant samples: GM-CSF, IFN-γ, IL-10, IL-17A, RANTES and TNF-α. The same steps were followed for the preparation of the samples and reagents. The standards that were used are indicated in table 2.7.

2.14.2.1 Preparation of reagents for immunoassay

Preparation of Antibody-immobilized Beads

Individual bead vials were used. Each antibody-bead vial was sonicated (Bransonic Ultrasonic bath sonicator) (Sigma-Aldrich, South Africa) for 30 seconds and then vortexed (Stuart® equipment, UK) for 1 minute. Eighty µL of each antibody-bead vial was added to the mixing bottle and then to bring to a final volume of 5.6mL, bead diluent was added. All the other reagents were prepared as described above.

Table 2.7: Preparation of Human cytokine standard and working standards

Standard concentration (pg/mL)

Volume of Deionized Water to add

Volume of Standard to add

10,000 250µL 0

Standard concentration (pg/mL)

Volume of Assay Buffer to add

Volume of Standard to add

2000 200µL 50µL of 10 000pg/mL

400 200µL 50µL of 2000pg/mL

80 200µL 50µL of 400pg/mL

16 200µL 50µL of 80pg/mL

3.2 200µL 50µL of 16pg/mL

The assay procedure that was used for Human Cytokine/Chemokine magnetic bead kit (EMD Millipore, USA) were also used for this kit. The overview of the assay is given in figure 2.12. These plates were run on Luminex MAGPIX® (Luminex Xmap technology, Bio-rad, USA) with the program, Bioplex manager. Calibration and verification was done using the Calibration and performance verification kit (MAGpix, Bio-rad, USA).

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Figure 2.13: The 96-well plate layout of plate 1 (A) and plate 2 (B) for the Human Cytokine/Chemokine

magnetic bead assay. Standard 0 = blank, standard 3.2 = low concentration standard, standard 10 000= Highest concentration std, QC1 = Assay Quality control 1, QC2 = Assay Quality Control 2. Evaluate QC1 and QC2 as recommended by kit manufacturer and the interpolate control just like any other sample. Each circle represents a well. The samples was done in triplicates for each time point as well as for stimulations with and without hIL- 2.