Author: J.E. Colligan et al, Translator: Xuitao Cao et al, This experiment is from "Compendium Immunology Laboratory Guide".
Operation method
BrdU Detection of D and B Cell Proliferation Move basic program Material Laboratory Animals 0.8 mg/mL B r d U (Sigma) water solution (oral) or 4 mg/mL B r d U PBS solution (injection) PBS, p H 7.4 0.15mo l / L ice-cold NaCl Ice-cold 95% ethanol Paraformaldehyde Fixative D N A enzyme I solution Anti-B r d U - F I T C (Becton Dickinson Immimocytometry) 15m m X 75m m round bottom polystyrene tubes Flow cytometers capable of three- or four-color analysis 1 . Administer B r d U by drinking water (dissolve .8 m g / m l B r d U in sterile drinking water; because B r d U is light-sensitive, a freshly prepared aqueous solution should be changed daily) or by intravenous or intraperitoneal injection of a PBS solution of B r d U at a concentration of 4 m g / m l. Administer 0.2 ml (0.8 m g ) per mouse as described in Appendix 2E. If an accurate calculation of the time of initial administration is required, B r d U may be given by both injection and drinking water. 2 . Prepare a single-cell suspension from the lymphoid organ (Box 2.1). Immunofluorescence of the cells (Box 4.1). If the cells have been surface-labeled in a microtiter plate, transfer the cells to a 15 m m X 75 m m tube. 3 . Use appropriate antibodies and fluorescein for surface labeling of the cells. If the anti-B r d U -F I T C antibody recommended in this protocol is used, then do not use the F T T C cross-linked antibody for surface labeling. 4 . Add ImlPBS and centrifuge at 300 g for 6 min at 4°C. Discard all supernatant and suspend the cells in 0.5 ml of ice-cold 0.15 mol/LNACR. If propidium iodide is used to differentiate between live and dead cells (Section 4.1), wash (Steps 4 to 5) 4 or 5 times before fixation and punching to remove all extracellular propidium iodide. 5 . Gently shake the cells while adding 1.2 ml of ice-cold 95 % ethanol dropwise to avoid cell clustering. Let the cells stand in an ice bath for 30 m i n . Add 2 ml of PBS and centrifuge at 450 g at 4°C for 6 m i n . 6. Discard supernatant thoroughly. Suspend the cells with I ml of paraformaldehyde fixative. Allow to stand at room temperature for 30 min, centrifuge at 4°C for 6 min. 7 . Discard the supernatant. Suspend the cells with l ml of DNAase I solution. Allow to stand at room temperature for 10 millimeters. Add 2 ml of PBS and centrifuge at 450 g at 4°C for 6 m i n . 8 . Discard supernatant. Suspend the cells and incubate for 30 m i n m i n t e r m s with 10 M 1 anti-BRDU-FITC antibody at room temperature. Add 2 ml of PBS and centrifuge at 450 g at 4°C for 6 mln. 9. Incubate with 500M1 PBS. Suspend the cells with 500M1 PBS. Perform flow cytometric analysis immediately (Unit 4.2) or store at 4°C under light for more than one week. 2 ) or store at 4°C under light for no more than one week. For more product details, please visit Aladdin Scientific website.