Cd19 apc clone sj25c1

Fluorochrome-conjugated monoclonal antibodies (mAbs) used included FITC-Mouse-IgG1 (clone X40), PE-Mouse-IgG1 (clone MOPC-21), APC-Mouse-IgG1 (clone SJ25C1), PerCP-Mouse-IgG1 (clone X40), APC-CD25 (clone 2A3), PerCP-CD4 (clone SK3), FITC-CD8 (clone 2D1), CD19-APC (clone SJ25C1), HLA-DR (clone L243), CD38-APC (clone HB7), CD3-FITC (clone SK7), CD16 + 56-PE (clone B73.1), CD45-PerCP (clone 2D1), CD4-FITC (clone SK3), CD8-PE (clone SK1) (BD Biosciences, San Jose, CA).

PBMC isolated by Ficoll gradient were incubated with pronase during 20 minutes in a 37°C water bath. Neat and 1/160 diluted serum was added to pronase-treated PBMC (Sigma Aldrich, St. Louis, MO) and the mix was incubated during 30 minutes at room temperature. Anti-CD3 Pacific Blue (clone UCHT-1, Immunostep, Salamanca, SPAIN), CD19 APC (clone SJ25C1, BD Biosciences, San Jose, CA) and subsequently Fab´-IgG FITC (Dako, Glostrup, DENMARK) were added and samples were acquired using a FACS-Canto II (BD Biosciences). Flow cytometry crossmatch for class-I antigens was considered positive in the CD3+ gate when the ratio: median fluorescence value Serum / median fluorescence value Negative Control was > 1.5; for class-II antigens, the FCXM was considered positive in CD19+ gate when the ratio was > 2.0.

In the FASCIA method, diluted whole blood is stimulated for seven days with three mitogens ConA, phytohemagglutinin (PHA), and pokeweed mitogen (PWM) as described in detail before [8 (link)]. Minimal blood volume requirement for the test is 0.5 ml. We used the following mitogens and final concentrations in RPMI media (supplemented with 2 mM L-glutamine, HEPES, 100 IU/ml penicillin, and 100 IU/ml streptomycin): PHA 10 μg/ml, ConA 10 μg/ml, and PWM 5 μg/ml (all mitogens from Sigma-Aldrich, USA). PHA and ConA are primarily T cell mitogens, and PWM is widely used to stimulate B cells. Our laboratory has tested that samples can be stimulated after approximately 27 h storage or transport at room temperature without compromising the results (data not shown).
After the incubation, red cells were lysed with IOTest 3 Lysing Solution (Beckman Coulter, USA) and stained with following directly conjugated antibodies: CD8 PerCP-Cy5.5 (clone SK1), CD19 APC (clone SJ25C1), and BD Simultest CD3-FITC/CD4-PE (clones SK7 and SK3 respectively), all antibodies from BD Biosciences, USA. Flow cytometry data was acquired with FACS Canto equipment (BD Biosciences), and the data analyzed with FACS Diva software (BD Biosciences).