Evaluating the Efficacy of Subcellular Fractionation of Blast Cells Using Live Cell Labeling and 2D DIGE - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 321

2. Materials

All solutions should be prepared using high-purity water (resistivity ³
18.2 M W, TOC £ 1 ppb, at 25°C). Reagents are analytical grade
unless specifi ed otherwise. Prepare all cell-culture-related reagents
in laminar fl ow hood for the purpose of sterility. Prior to use, ensure
all buffers are thawed and mixed well by vortexing. In the provided
example, immature blast cells are initially isolated from bone mar-
row by removal of CD 3+ T-cell and CD 19+ B-cell populations, as
blast cells are a clinically relevant cell type for investigating a
patient's response to chemotherapy. This step is not specifi cally
required for the combined LCL/2D DIGE approach.

2.1. Magnetic
Cell Sorting

1. Culture media: Iscove's Modifi ed Dulbecco's Medium
(IMDM) (Invitrogen, Carlsbad, CA, USA) supplemented with
20% (v/v) fetal calf serum (FCS), penicillin/streptomycin
antibiotics, L -glutamine, and DNase I. Bring media to 37°C
before use by incubating in a 37°C water bath. Use DNase I at
2,500 U/50 mL.
2. Trypan blue solution (Sigma-Aldrich, St. Louis, MO, USA).
3. Phosphate-buffered saline (PBS): To prepare 1 L of 20×
solution, weigh 160 g of sodium chloride, 4 g of potassium
chloride, 4 g of potassium dihydrogen phosphate, and 23 g of
disodium hydrogen phosphate and transfer to a 2-L conical
fl ask containing ~800 mL of ultrapure water. Mix for 20 min.
Make up the volume to 1 L with water and adjust pH to pH
7.4. Autoclave the solution prior to use.
4. MACS buffer: Add 2.5 mL of MACS BSA (Miltenyi Biotec
GmbH, Bergisch Galdbach, Germany) to 1,450 mL of
autoMACS rinsing solution (Miltenyi Biotec GmbH). Keep
buffer at 4°C.
5. MicroBeads conjugated to monoclonal anti-human CD3
antibodies (Miltenyi Biotec GmbH). CD3 MicroBeads are
supplied as a suspension containing stabilizer and 0.05% sodium
azide. Store protected from light at 4°C.
6. LD column (Miltenyi Biotec GmbH).
7. Pre-separation fi lters (Miltenyi Biotec GmbH).
8. Column adaptor (Miltenyi Biotec GmbH), required to insert
column into the MidiMACS separator.
9. MicroBeads conjugated to monoclonal anti-human CD19
antibodies (Miltenyi Biotec GmbH). CD19 MicroBeads are
supplied in buffer containing stabilizer and 0.05% sodium
azide. Store protected from light at 4°C.
10. 50-mL Falcon polypropylene conical centrifuge tube (BD,
Franklin Lakes, NJ, USA) (see Note 2).