Biomedical Engineering Reference
In-Depth Information
is used because it is cell impermeable, allowing for the selective
measurement of cell surface/extracellular
-lac [
6
]. The substrate
conversion is monitored over time, where the slope of the reaction
(i.e., initial velocity) is directly proportional to surface expression.
This is unlike endpoint measurements that are often used in other
surface expression assays (ELISA, fl ow cytometry). We described
here the transfection of
ʲ
ʲ
-lac fusion protein constructs and the pro-
cedure of the
-lac assay in a 48-well format. The assay is performed
with live adhered cells and can be scaled down (96 or 384-well
plate) for use in higher-throughput applications.
ʲ
2
Materials
Prepare all solutions using ultrapure water (Milli-Q) and analytical
grade reagents. All concentrations are fi nal concentrations unless
stated otherwise. Solutions are stored at room temperature unless
stated otherwise.
1. Phosphate buffered saline (PBS): NaCl 137 mM, KCl 2.7 mM,
Na
2
HPO
4
10 mM, KH
2
PO
4
1.8 mM, pH 7.4 is used to wash
the cells and dilute the substrate nitrocefi n to the working
concentration.
2. Dulbecco's modifi ed Eagles medium (DMEM) (Wisent):
Supplement with 10 % (v/v) fetal bovine serum (FBS) (Sigma).
100 U/mL Penicillin, 100
2.1 Reagents
ʼ
g/mL Streptomycin. G418
(500
g/mL) (Bioshop)
are added as selection agents for the creation of stable cell lines
expressing the
ʼ
g/mL) (Bioshop) or Puromycin (1
ʼ
-lac constructs of interest.
3. Poly-
D
-lysine: Dissolve 5 mg poly-
D
-lysine hydrobromide
(mol. Wt. 30,000-70,000) (Sigma) in 50 mL of sterile ddH
2
O
to obtain a 0.1 mg/mL solution and store at 4 °C.
4. Nitrocefi n: Dissolve 5.16 mg of nitrocefi n (Calbiochem) in
1 mL dimethyl sulfoxide (DMSO) to obtain a 10 mM stock
solution (100×) and store in 100
ʲ
L aliquots at −80 °C.
5. Trypsin/EDTA: 0.25 % w/v trypsin, 0.53 mm EDTA (Wisent)
is diluted to 0.05 % trypsin, 0.10 mM EDTA with PBS. Store
at −20 °C until use.
6. Polyethylenimine (PEI) (Polysciences). Dissolve PEI at 1 mg/
mL in ddH
2
O, adjust to pH 7 with 0.5 N HCl, fi lter-sterilize,
and store at −80 °C.
7. Human embryonic kidney (HEK) 293 cells (ATCC
CRL-1573).
8. A hemocytometer (Fischer Scientifi c) is used to count cells.
9. Cellstar clear 48-well plates (Greiner Bio-One) are utilized to
conduct the
ʼ
ʲ
-lac assay.
