Biomedical Engineering Reference
In-Depth Information
10 % glycerol were supplemented with 10
ʼ
g/ml aprotinin,
g/ml pepstatin, 1 mM PMSF, 1 mM
Na
3
VO
4
, and 10 mM NaF prior to cell lysis.
9. Protein assay: BCA protein assay kit (Pierce; cat# 23227) was
used to determine protein concentration in cell lysates.
10. QuantaRed Enhanced Chemifl uorescent HRP Substrate
(Thermo Fisher Scientifi c; cat # 15159): QuantaRed working
solution was prepared by mixing QuantaRed Enhancer
Solution, QuantaRed Stable Peroxide, and QuantaRed ADHP
at 50:50:1 immediately before use. 100
10
ʼ
g/ml leupeptin, 10
ʼ
l of QuantaRed work-
ing solution was added to each well and incubated for 15 min
at room temperature. The reaction was stopped with 10
ʼ
l of
QuantaRed Stop solution per well and the plate mixed for
10-30 s. Plates were either immediately analyzed on a micro-
plate reader or stored for later analysis at 4 °C for several days
wrapped in foil with minimal effect on fl uorescence intensity.
11. Chemiluminescence detection: ChemiGlow West Chemilu-
minescence Substrate Kit (ProteinSimple; cat # 60-12596-00)
was used to visualize positive signals in immunoblots.
12. Instruments: Fluorescence was measured using a multimode
microplate reader POLARstar Omega instrument (BMG
LABTECH). For fl ow cytometry assay, BD FACSCanto™
instrument was used. Chemiluminescence was detected using
a CCD camera (Alpha Innotech). Plates were routinely spun in
a tabletop centrifuge at 2,000 rpm (~500 ×
g
, Eppendorf 5416)
prior to reading.
13. 96-well plates: Standard tissue culture-treated 10 cm, 5 cm,
6-well, and 96-well plates were used for cell culture work
(Corning). siRNAs were aliquoted and pooled in Microseal
PCR working plates (Bio-Rad #MSS96011) and plates were
sealed with Microseal “B” adhesive seal (Bio-Rad; MSB 1001).
Cell transfection, HGF treatment, and MET staining and visu-
alization were performed using solid black 96-well cell culture
plates (Costar; cat # 3916). Cell staining and reading for fl ow
cytometry was performed using round-bottom 96-well poly-
propylene microplates (Corning, cat# 3355).
ʼ
2.3 siRNA Library:
Handling and Storage
1. Ubiquitin siRNA library (0.25 nmol siRNA/well; QIAGEN
cat. No. 1027412; SO 0140368892) comprises 129 genes dis-
tributed over six 96-well plates (named Ubi01-Ubi06). The
library includes four different targeting siRNAs for each of 89
genes or three different targeting siRNAs for 40 genes. Each
plate carried siRNAs for 24 genes and eight control siRNAs
(Ubi01-Ubi05) with the exception of Ubi06, which carried
siRNAs for nine genes and ten control siRNAs.
2. Prior to resuspension, the master siRNA plates were centri-
fuged at 500 ×
g
for 5 min.
