Ki 67 fitc antibody

For intracellular Ki67 staining, cells were fixed for 3 min with Fixation Buffer (Fix & Perm, Life Technologies) before adding pre-cooled 50% methanol for 10 min at 4°C. Cells were then washed in PBS with 5% FBS and incubated for 30 min with the Ki-67 FITC antibody diluted in permeabilitzation buffer (1:10; clone B56, BD Biosciences). For cell cycle analysis, cell were suspended in 0.03% saponin (Sigma-Aldrich) in PBS and then incubated in 20 mM 7-aminoactinomycin D (7AAD; Sigma-Aldrich) for 30 min at room temperature in the dark, followed by 5 min at 4°C. Then, Pyronin Y (Sigma-Aldrich) was added at a final concentration of 1.5 μg/ml and cells were further incubated at 4°C for 15 min. Flow cytometry was performed in a LSRII flow cytometer (BD Biosciences). The data were analyzed using the FlowJo software (BD Biosciences). To correct the overestimation of G2/M population by miss discrimination of cellular doublets, FL2W versus FL2A of the 7AAD dye was plotted before gating for the distinct cell cycle phases [51 (link)].

The presence of quiescent cells was determined by staining the suspension cells and the CAFC with CD45-PC7 for 30 min on ice followed by fixation with 1% paraformaldehyde, then incubated for 15 min at room temperature with permeabilization buffer (BD Bioscience). Cells were then incubated on ice for 45 min with Ki-67-FITC antibody (BD Bioscience) washed twice in PBS containing 2% FBS and resuspended in the same buffer containing 7-AAD.and analyzed by flow cytometry. For the proliferation assay, irradiated MS-5 cells were added to the upper chamber of a 1 µm transwell insert and allowed to adhere overnight. Primary MCL cells depleted of unwanted cells by magnetic beads were added to the upper insert and maintained for 4 weeks for CA formation. At week 5, 10 µM of BrdU was added to both chambers of the transwell plate and incubated for 16 hrs. The CD19+CD133 and CD19−CD133+ MCL subpopulations were processed as described in section “Cell depletion and MCL fractionation”. BrdU incorporation into the CD19+CD133 and CD19−CD133+ subpopulations was determined using the BrdU Cell Proliferation Assay Kit (Cell Signaling). After staining, the cells were analyzed by flow cytometry.