Antibodies and reagents
Phycoerythrin (PE) conjugated anti-IgD, anti-CD3 and isotype control, IgG1, were from Becton Dickinson (Mississauga, ON). Tri-color (TC) conjugated anti-CD19 was from Caltag (Burlingame, AL). FITC conjugated anti-CD38 was from Abcam (Cambridge, UK). RosetteSep human B lymphocyte enrichment cocktail was purchased from StemCell Technologies Inc. (Vancouver, BC). Sheep RBCs were purchased from Cedarlane Laboratories Ltd (Hornby, ON).
Tonsils
Tonsils were obtained from the Alberta Children's Hospital with ethical approval. Cell suspensions were prepared by cutting the tissue into fine pieces using scissors or razor blades in RPMI 1640 (Gibco Laboratories, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) (Gibco). The tonsil tissue fragments were then forced through a nylon screen of 70 μm mesh size to disrupt the pieces into single cells. Cells were then washed twice with RPMI/FBS and counted.
Preparation of human red blood cells
Human blood was collected from healthy donors into heparin vacutainers. Mononuclear cells were removed by diluting the whole blood with an equal volume of RPMI/FBS, layering over lymphocyte separation medium (MP Biomedicals LLC, Solon, OH), and then centrifugation at 1160 × g for 20 minutes with no brake. Cells at the interphase were aspirated and discarded, while the loosely pelleted RBCs were washed and returned to their original volume using Alsever's solution. Prepared RBCs were counted and used for up to one month. All steps were performed at room temperature.
Purification of tonsil B lymphocytes
To purify B lymphocytes, 2 × 108 tonsil cells were mixed with a 50-fold excess (1010) of human or sheep RBCs. The cells were centrifuged at 350 × g for 5 minutes and then resuspended in 4 mL of RPMI/FBS. 200 μl of RosetteSep was then added; the sample was gently mixed by rocking and incubated for 20 minutes. The sample was then diluted with an equal volume of phosphate buffered saline (PBS) containing 2% FBS and 1 mM EDTA. Lymphocyte separation medium was carefully layered under the preparation and centrifuged for 20 minutes at 1160 × g with no brake. Cells at the interface were collected, washed and saved for analysis. All steps were performed at room temperature.
Reverse transcription PCR
Up to 107 cells were used to prepare total RNA using the RNeasy mini kit (Qiagen, Valencia, CA). 5 μg of total RNA was reverse transcribed using Superscript III (Invitrogen, Carlsbad, CA) with oligo dT primers. Polymerase chain reaction (PCR) was performed in 25 μl volumes with TAQ PCR master mix (Qiagen, Valencia, CA) using 35 cycles of 1 min at 95°C, 1 min at 58°C, and 50 sec at 72°C. Primers for CD20: forward 5' GCA GCA ACG GAG AAA AAC TC 3' and reverse 5' GAA GAA GCG TGA CAA CAC AAG 3'. Primers for CD3: forward 5' CTG GGA AGT AAT GCC AAG GA 3' and reverse 5' TAG ACC CCA ACA GCA AGG AC 3'. Primers for Beta actin: forward 5' CAC TCT TCC AGC CTT CCT TCC 3' and reverse 5' GTG TTG GCG TAC AGG TCT TTG 3'. PCR products were resolved on 1% agarose Tris-Borate-EDTA (TBE) gels with ethidium bromide and photographed to observe relative signals of each amplified transcript.
Flow cytometry
Cells were incubated with directly conjugated antibodies for 15 min at room temperature, washed and acquired on a flow cytometer (FACScan, Becton Dickinson). The data were analyzed using the FlowJo program (Tree Star Inc, San Carlo, CA).