By using a blood cell separator (Spectra v 6.1, Cobe, USA), (2 4) × 109 PBMC cells from each healthy volunteer were obtained in a total volume of 50 mL.
Briefly, peripheral blood mononuclear cells (PBMCs) were collected using a blood cell separator (Haemonetics CCS, Haemonetics Corporation, Braintree, MA, USA) and stimulated with human recombinant interleukin (IL -2 (rIL -2200 U/ml; Primmune Inc. Kobe, Japan) and 5 μg/ml antibody to CD3 (MACS GMP CD3 pure; Miltenyi Biotec Inc. Auburn, CA, USA).
Then, we determined the erythrocyte density of the cell suspension using a blood cell-counter.
Along the way Poonawalla got his Ph.D. in a novel technology that uses a blood-cell separator to extract serum without harming the horse.
In experiments where ATC were irradiated, cells received a single 2500 cGy dose using a blood bank cell irradiator (Nordion, Ottawa, Canada) to prevent lymphocyte proliferation [ 10] and determine whether cytotoxic activity of BiAb-armed ATC is radioresistant.
Briefly, RBcs-treated DCs or control DCs were washed extensively and then purified with microbeads on auto-MACS columns using a Blood Dendritic Cell Isolation kit (Miltenyi Biotech, BergischGladbach, Germany) according to the manufacturer's instructions.
We extracted DNA for sexing from the frozen blood cells using a Blood Mini kit (A&A Biotechnology, Gdynia, Poland).
Blood samples were collected from the proband (II-2) and her family members (II-1, III-2, IV-1, and IV-2; Figure 1), and genomic DNA was isolated from the peripheral white blood cells using a blood DNA isolation kit (NucleoSpin Blood XL; Macherey Nagel, Germany).
Genomic DNA was isolated from Jurkat, U937, Bv173, K-562, OC1-AML3, KG-1A and RPMI 8226 cell lines, peripheral blood samples, and bone marrow aspirates using a Blood & Cell Culture DNA Mini Kit from Qiagen GmbH (Hilden, Germany) and following the manufacturer's instructions.
Red blood cells were lyzed for 90 seconds using a red blood cell lysing buffer (Sigma Aldrich, Vienna, Austria).
Genomic DNA was extracted from whole blood using a Blood & Cell Culture DNA kit (QIAGEN).
