Sanquin Diagnostiek B.V.

Bloodtype genotyping (including allelic discriminations, clotting diseases and red cell deficiencies)

Leukemia markers/ gene expression (eg, p190, bcr, braf, myd88 etc)

HLA typing (low- and high resolution using NGS)

Nucleic extractions (DNA and RNA)

Nucleic extractions (DNA and RNA)

We can perform proliferation assays based on 3H thymidine incorporation and/or proliferation dye.

Description of the assay:
1. Effector cells (E):
a. positive (CD56+) selection of NK cells from fresh blood-isolated peripheral mononuclear blood cells (PBMC) using magnetic beads– preferred method of selection
b. negative (all other cells but NK cells depleted from PBMC)
2. Target cells (T):
a. SKBR3 breast carcinoma cell line, positive for Her2/Neu, possible to opsonize with trastuzumab to perform antibody-dependent cellular cytotoxicity assay (ADCC)
b. A431 colon carcinoma cell line, positive for EGFR, possible to opsonize with cetuximab to perform ADCC
c. Raji Burkitt lymphoma cell line, positive for CD20, possible to opsonize with rituximab to perfrom ADCC
3. Assay: target cells are labeled with radioactive 51Cr , and level of release is measured after incubation with NK cells, assay is performed in technical triplicate

General design of the assay:
- Target cell lines of interest tested (can test all three cell lines, depending on the final design either all target cell lines in parallel or separately)
- Test both spontaneous and ADCC (ADCC is about 2-fold higher than spontaneous cytotoxicity)
- Test 6 different healthy individuals
- Test different E:T ratio
- Step 1: test one concentration of all n=x compounds in these conditions
- Step 2: depending on the result, choose fixed E:T ratio and perform dose-dependency with n=x compounds

Additional assays/ expertise:
Besides the NK cell cytotoxicity assays, we have several assay set-ups available to determine activation, proliferation and differentiation of immune cells, mainly using flow cytometry as a read-out. We generally focus on complete peripheral blood mononuclear cells or isolated T cells for these type of analyses, but these can of course also be applied to NK cells.

Biomarkers Services

Biology Services

Clinical Trials, Consulting, and Management Services

Pharmacology & Toxicology Services

Clinical Immunology Tests Services

Blood Banking and Transfusion Medicine Services

Clinical Hematology Services

Clinical Laboratory Services

Clinical Research Services

Immunoassays Services

Bioanalytical Assays Services

eg. Selection CD8+ T-cell epitopes, antigen-specific cytotoxic T-cell monitoring and On/ off target screening.

Antibody Services

Cell Viability & Proliferation Assays Services

Testing of Cell Lines Services

Cells and Tissues Services

Protein Quantification Services

Protein Purification and Quantification Services

Protein Services

Biochemistry & Molecular Biology Services

Immune Cell Assays Services

Functional & Cell Type Specific Assays Services

Gene Panel Testing Services

Clinical Molecular Diagnostics Services

RNA Services

DNA Extraction and Purification Services

DNA Services

Nucleic Acid Services

Mesenteric vessel staining

Isolate, fix and staining of vessels

Migration- and Transmigration assays

Amongst our validated markers: C1INH (including inhibitor activity, Ag conc, Ab conc, Ab neutralising capacity), C1q, C3, C3d, C4, sC5b-9, C5, CH50, AP50, LP act, cytokines. In addition, we deliver the advantage to combine studies with our capailities in cellular assays (eg investigating Th/ Treg-cells responses).

Cell Permeability Assay - leakage assay

Day 1
Seed epithelial cell line H292 in RPMI / 10%FCS / PS / L-glut
12WP transwell membrane
0.4 μm pore
12mm membrane
8.0•104 / well
RPMI / 10% FCS / 1% L-glutamin

Note/ It is not possible to view the forming monolayer through the membrane; therefore, seed a similar amount of cells in a 24WP for comparison. The 12mm TW has a growth area of 1cm2; the wells from a 24WP have a growth area of 2cm2; seed two wells of a 24WP with 8.0•104 / well and 2 wells of a 24WP with 16•104 / well

Day 2 / 3
Refresh medium

Day 4
Remove phonol red:
Wash cells and bottom of TW with
Hepes containing 0.5% CeAlb
Transfer TW to new/clean 12WP

Add Dextran-Fluorescein 3000 and / or drugs
Allow dextrin-fluorescein to pass through membrane for 2 hrs
Measure λex 494 nm / λem 521 nm on an elisa plate reader
Magellan Fluorescein Standard: λex 485 nm / λem 535 nm

Cell Invasion & Migration Assays Services

Drug Discovery & Development Services

Cell Death Assays Services

Pharmacology Services