Microsoft word - detection of lacz expression by facs.doc

Detection of LacZ expression by FACS-Gal analysis

Procedure

1. Prepare single cell suspension.
a) Harvest cells.
b) Filter the suspended cells through 30µm cell strainer while keeping cells on ice.
c) Count the cell concentration.
d) Centrifuge the filtered cells at 1,200rpm for 4 min.
e) Resuspend the cells with HBSS+ staining buffer (HBSS+, ca. 2×107 cells/ml if
2. Load FDG into cells. a) Aliquot 100µl suspended cells for staining. Prepare controls including unstained control, single color controls for FACS compensation. b) Prepare 2mM FDG working solution by diluting 20mM FDG stock solution with ice-cold distilled water. Prepare working FDG control solution by diluting FDG control stock solution by 1:10. Aliquote 100µL FDG working solution or control solution to amber tubes. c) Prepare 15ml tubes with 2ml HBSS+ buffer and keep on ice. d) Make sure all the sets of cell sample tubes, FDG tubes and 2ml HBSS+ tubes are e) Prewarm cell sample tubes and corresponding FDG (or control) tubes in 37ºC water bath for 10min. 2ml HBSS+ tubes are kept on ice during loading. f) Follow the FDG loading strategy (see below) to load FDG into cells. Transfer prewarmed cells into corresponding FDG tubes. Mix thoroughly. Return to 37ºC bath for exactly 1 min. Stop the FDG loading at the end of one min by transferring mixture into 2ml ice-cold HBSS+. g) Keep on ice for 1.5 hours to allow accumulation of FITC release from FDG in h) Centrifuge at 1500rpm for 7 minutes, discard supernatant and resuspend cell pellets 3. Stain cells with antibodies. a) Add 2µl antibodies to cells and slowly rock at 4ºC for 15 min. b) Centrifuge at 1,200rpm for 4 min., discard supernatants and resuspend cell pellets c) Add 7µl 7-AAD solution to 200µl cells 4. Perform FACS analysis. a) Adjust voltage. b) Run compensation. c) Run samples and collects FACS data. d) Analyze your FACS data. Materials and Reagents

1. 30µm cell strainer
2. Amber eppendorf tubes (USA scientific)
3. 20mM FDG stock solution (Fluorescein di[β-D-galactopyranoside], Sigma, F2756)
Dissolve FDG in DMSO and ethanol solution (DMSO:ethanol=1:1) completely, and
then gradually add ice-cold water (DMSO:ethanol:water=1:1:8) into FDG drop by drop. Aliquot and keep the FDG solution at -20ºC for long-term storage. Make FDG diluent (DMSO:ethanol:water=1:1:8) as FDG control stock solution. Dilute 1:10 to make a working FDG solution or FDG control solution when needed. 4. HBSS+ buffer, ice-cold (1x HBSS, 2% Fetal calf serum, 10mM Hepes buffer pH 5. Fluorescence-conjugated antibodies
7. 7-AAD solution (BD Pharmingen)
Equipment

Water bath at 37ºC
BD FACScan, Calibur, Canto or LSR analyzer
Troubleshooting

1. Weak LacZ+ cell signal. Possible reasons:
a) Make sure that the volume ratio of cell to FDG is exactly 1:1.
b) Make sure to follow the FDG loading strategy.
c) The cells have to be kept on ice after FDG loading. Each step should be performed
on ice. Even when the FDG-loaded cells are carried around, they have to be on ice.
Critical Steps

Step 1.e. Take care to remove any remaining harvesting medium or buffer in the tube.
Single cell suspension has to be made in HBSS+ buffer, which has been tested to work best for FDG loading. Step 2.c-e. To make sure to incubate cell and FDG mixtures for exactly 1 minute, the FDG loading strategy in Figure 1 should be used.
Step 2e. From this step downward, all samples needs to be kept on ice at all.
Keywords

Flow cytometry, galactosidase, Rosa26-LacZ, FDG, FACS-Gal
FDG loading strategy
1. Make sure all tubes (FDG/control and 2ml HBSS+) are properly prepared before loading. Note that the 2ml HBSS+ tubes are kept on ice during loading. 2. Place paired tubes of FDG/Control and cells (maximum of four pairs in each group) simultaneously into a 37°C water bath at 3-minute intervals.
3. After 10 minute of warming, load FDG into cells as shown in the figure below:
a) Set timer at 0'00'' and start counting up. Take the first paired tube of cells and
transfer cells into the corresponding FDG tube. Mix well. Place the FDG and cell
mixture tube back into the 37°C water bath exactly at each 20 second mark.
b) Transfer the mixtures into the corresponding 2ml HBSS+ (on ice) exactly at the
end of each tube's 1 minute incubation time.
4. After finishing the group, the next group will be ready for mixing and incubation in
20 seconds. When the next group is ready, repeat loading steps 2a,b.

References:

1. Guo W. et al. Multi-genetic events collaboratively contribute to Pten-null
leukaemia stem-cell formation. Nature 453, 22: 529-534. 2. Soriano, P. Generalized lacZ expression with the ROSA26 Cre reporter strain. 3. Nolan, G.P. et al. Fluorescence-Activated Cell Analysis and Sorting of Viable Mammalian Cells Based on ß -D-galactosidase Activity after Transduction of Escherichia coli lacZ. PNAS 85, 2603-2607 (1988). 4. Rotman, B. et al. Fluorogenic Substrates for ß-D-galactosidases and Phosphatases Derived from Fluorescein (3, 6-dihydroxyfluoran) and Its Monomethyl Ether. PNAS 50, 1-6 (1963).

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