A detailed Labeling Protocol follows. In summary, the biotin-conjugated antibodies simultaneously stain the lineage-committed cells according to their different specificities. Two microliters of each pre-diluted antibody conjugate is sufficient to stain 10^6 cells in 10 µl of staining buffer. Negative selection is then performed to enrich for uncommitted hematopoietic progenitors. The Mouse Lineage Panel can be used with similar efficiency with magnetic particle separation systems or flow cytometric cell sorting. For convenience, all antibodies have been optimized and pre-diluted to provide maximum efficiency for cell separation. The antibodies are provided in separate vials to assure product stability and to allow flexibility of separation protocols.
Hematopoietic progenitor cells separated after staining with the Mouse Lineage Panel have been tested in flow cytometric analysis, colony assays in methylcellulose, differential staining microscopy, immunoprecipitation, and western blot analysis. Use of the Mouse Lineage Panel does not alter the morphology, phenotype, or function of the separated cells in such assays.
LABELING PROTOCOL FOR ENRICHMENT OF HEMATOPOIETIC PROGENITOR CELLS
1. Prepare a single-cell suspension from bone marrow or other hematopoietic organ.
Notes:
-The femurs and tibiae of one adult mouse typically yield 20 -60 × 10^6 hematopoietic cells. Two mice will yield 0.5 -1.0 × 10^6 lineage-negative cells.
-The presence of red blood cells (RBCs) does not affect the staining with the Mouse Lineage Panel. We have observed that initial lysis of RBCs, using buffered ammonium chloride, reduces the effectiveness of the staining. Cell separation by gradient may be more appropriate. If RBC lysis is required for further analysis, it should be performed after the separation procedure.
-If the separated cells are to be used for culture or in vivo assays, aseptic conditions and sterile media/buffers must be used.
2. Count the cells, and resuspend them in cell-staining buffer [eg, BD Pharmingen™ Stain Buffer (FBS), Cat. No. 554656] at a final density of 100 × 10^6 cells per ml.
3. Set aside a sample of unstained cells (~5 × 10^6 cells) to be used in the flow cytometric analysis in Step 7.
4. Add the biotinylated antibodies from the Mouse Lineage Panel, using 2 µl of each prediluted antibody per 10^6 cells, and incubate on ice for 15 minutes.
5. Wash the stained cells twice, using the buffer recommended for the cell-separation system to be used.
6. Resuspend the cells at a final density of 20 - 100 × 10^6 cells per ml. The stained cells can be used in various cell-separation systems.
Notes:
-For each separation method, the recommended protocol described by the manufacturer should be followed.
-For flow cytometric cell sorting, the cells should be labeled with streptavidin (or avidin) bound to an appropriate fluorochrome.
-For immunomagnetic cell separation using a High Gradient Magnetic Separation Column, the cells should be labeled with the
appropriate streptavidin-bound magnetic particles (eg, BD™ IMag Streptavidin Particles Plus - MSC, Cat. No. 557811). For immunomagnetic cell separation using the BD IMag™ Cell Separation Magnet (Cat. No. 552311), we recommend the BD™ IMag Mouse Hematopoietic Progenitor Cell Enrichment Set - DM, Cat. No. 558451).
7. Samples of the total cell suspension, lineage-positive, and lineage-negative fractions should be analyzed by flow cytometry to evaluate the efficiency of the cell-separation procedure.
Note:
-The recommended concentration of the Mouse Lineage Panel antibodies is not saturating and allows the staining of the separated lineage-positive and lineage-negative fractions by the same monoclonal antibodies as used for the depletion.
