BD Biosciences
Technical Bulletin
January 2012
Technique for Loading Cells with BD Horizon™ Violet Proliferation Dye 450 (VPD450)
Technique for Loading Cells with
BD Horizon™ Violet Proliferation Dye 450
(VPD450)
Jeanne Elia, Monisha Sundarrajan, and David Ernst
BD Biosciences, San Diego
Technical Bulletin
Contents
1Introduction
3
Cell concentration
4
Dye concentration
7
Cell loading
8
Dye effects on the cell population
9
Effect of dye on cell signaling events
10Controls
10Conclusions
11References
Introduction
Fluorescein diacetate (FDA) and related molecules like carboxyfluorescein
succinimidyl ester (CFSE) are non-fluorescent molecules that diffuse into cells.
Once inside cells, they are hydrolyzed by intracellular non-specific esterases and
covalently bind to cellular components to become fluorescent products. Dead
cells and cells without intact membranes do not label with these dyes and are not
fluorescent. The BD Horizon™ Violet Proliferation Dye 450 (VPD450) is
functionally similar to CFSE since they contain both an esterase-cleavable group
and an amine-reactive succinimidyl ester group. VPD450 is particularly suitable
for multicolor applications where either green fluorescent proteins or FITClabeled antibodies are used. The VPD450 dye is excited by the violet laser
compared to CFSE and its fluorescein analogs which utilize the same lasers and
detectors as GFP, FITC, and Alexa Fluor® 488.
FDA dyes have multiple uses which include in vitro or in vivo cell tracking
experiments, fluorescent barcoding, and cell proliferation. In this set of
experiments, we have optimized the use of VPD450 for cell proliferation studies.
Stimulated cells labeled with VPD450 divide and form new generations of cells.
Each new generation of cells has approximately half the fluorescence intensity of
the previous generation. These generations of divided cells can be counted by
displaying a fluorescence histogram of the V450 channel. The VPD450 dye is
excited by the violet laser (emitting at 450 nm in the V450 channel), allowing for
multicolor staining with additional markers using the standard blue or red lasers.
Combining cell generation analysis with specific cell surface and intracellular
markers allows each investigator to determine when key events are taking place
in their experimental systems.
When loading cells with the VPD450 dye, there are three key details to have
successful labeling: cell concentration, dye concentration, and the physical
technique of loading the cells. In this technical note, we address these key
considerations and demonstrate the effects of the VPD450 dye labeling on the
ability of cells to proliferate or respond to activation signals using techniques
such as BrdU incorporation, cytokine production, and cell signaling.
BD Biosciences
Technical Bulletin
January 2012
Technique for Loading Cells with BD Horizon™ Violet Proliferation Dye 450 (VPD450)
To address each of these conditions, we analyzed both mouse splenocytes and
human peripheral blood mononuclear cells (PBMCs). Mouse splenocytes were
isolated, made into single-cell suspensions, followed by red blood cell lysis. In
the case of human samples, the blood was collected in heparinized tubes, then
separated using Ficoll-Paque™ density centrifugation to isolate the PBMCs. All
groups were washed twice in 1X Dulbecco’s phosphate buffered saline (PBS) to
remove any residual proteins from the wash buffers. The cells were transferred
into 15-mL polypropylene centrifuge tubes and labeled with VPD450. Optimal
conditions were determined for cell concentration, dye concentration, and loading
technique. In each experiment, a no-load VPD450 group was set up as a control.
Cells were activated in 6-well plates with either phytohemagglutinin (PHA) or
anti-CD3ε/CD28 antibodies (PBMCs) or with anti-CD3ε/CD28 antibodies
(mouse spleen) for 2–5 days. Cell proliferation was then assessed between
VPD450-loaded cells and the no-load (without VPD450) control cells. We further
chose to monitor proliferation in the same group of cells by traditional BrdU
incorporation using the BD Pharmingen™ APC BrdU Flow Kit (Cat. No.
552598). Thus, BrdU incorporation allowed us to compare the VPD450-loaded
samples to the no-load controls for their ability to synthesize DNA in response
to stimulation. The APC fluorochrome of the Anti-BrdU antibody (APC BrdU
Flow Kit; used for detecting incorporated BrdU) was excited by the red laser and
is detected in the APC channel. The 7-AAD (7-Aminoactinomycin D) dye from
the same kit was used for staining cellular DNA. It was excited by the blue laser
and detected in the PerCP channel. The VPD450 dye was excited using 405 nm
violet laser and detected in the BD Horizon™ V450 or Pacific Blue™ channel.
Data was collected using a BD™ LSR II flow cytometer and was then reanalyzed
using BD FACSDiva™ software.
BD Biosciences
Technical Bulletin
January 2012
Technique for Loading Cells with BD Horizon™ Violet Proliferation Dye 450 (VPD450)
Page 3
Cell concentration
I n order to find the optimal cell number for labeling with a fixed
1-μM concentration of VPD450 dye, we set up several experiments loading three
different concentrations of cells: either 1 x 106 cells/mL, 1 x 107 cells/mL, or 2 x
107 cells/mL. The intensity of the initial load was compared to the ability of cells
to undergo cell division (measured as number of VPD450 fluorescent peaks) after
three days in culture.
50
100
150
150
200
250
(x 1,000)
19%
104
P2
103
102
103
100
105
36%
P2
1 x 106 cells/mL
D
102
50
200
250
(x 1,000)
2 x 106 cells/mL
C
105
105
102
102
103
103
104
47%
P2
104
105
51%
P2
BrdU APC
1 x 107 cells/mL
B
104
No-load Cells
A
50
100
150
50
200
250
(x 1,000)
100
150
200
250
(x 1,000)
7-AAD
103
104
105
102
103
104
105
100 125
75
50
25
102
103
104
105
0
25
0
0
102
H
0 10 20 30 40 50 60 70 80 90
100
75
50
100
50
Count
G
150
F
150
E
102
103
104
105
VPD450
Figure 1. Human PBMCs at different cell concentrations were either loaded with 1 μM of VPD450
for 10 minutes at 37ºC or were left as untreated controls (no-load cells). 1 x 106 cells were plated
onto a 24-well plate coated with anti-CD3e + soluble anti-CD28 antibodies, then incubated
for three days. Cells were loaded with BrdU prior to harvest, then stained for incorporated
BrdU (APC BrdU) and DNA levels (7-AAD). Panel A represents cells only controls (no-load cells).
Cell concentrations varied from 1 x 107 cells/mL (B), 2 x 106 cells/mL (C), to 1 x 106 cells/mL (D)
when they were labeled with VPD450. Corresponding VPD450 histograms for the cultured cells
are shown in panels E–H. The 2 x 106 cells/mL concentration (C) displayed a decrease in BrdU
incorporation of 29%, while the 1 x 106 cells/mL concentration displayed a decrease in BrdU
incorporation of 63%. This data suggests that cell concentrations greater than 2 x 106 cells/mL
should be used for VPD450 labeling to observe expected responses in cell proliferation.
BD Biosciences
Technical Bulletin
January 2012
Technique for Loading Cells with BD Horizon™ Violet Proliferation Dye 450 (VPD450)
Dye concentration
Determining the optimal concentration of the VPD450 dye is also important
when setting up an experiment. It is always preferable to load with as little dye
as possible while still maintaining optimal peak brightness and resolution. Higher
concentrations of dye alter the ability of cells to respond to stimulation. The
following example is from human PBMCs that were loaded with varying
concentrations of VPD450 dye, and then stimulated for five days with PHA. Cell
aliquots were pulsed for 1 hour with BrdU, harvested, and then stained at days
3 and 5.
105
Day 5 No-load Cells
150 200 250
103
102
75 100
50
100
150 200 250
(x 1,000)
L
75 100 125
150
25 50
100
105
41%
P2
50
(x 1,000)
50
104
105
103
150 200 250
0
0
103
104
102
100
K
0 50 100 150 200 250 300 350
102
103
Day 5 10 µM VPD450
I
14.5%
P2
50
(x 1,000)
(x 1,000)
25
Day 5 1 µM VPD450
105
102
105
103
100
7-AAD
150 200 250
0
104
102
50
Count
103
104
105
103
104
6%
102
BrdU APC
P2
100
(x 1,000)
F
102
H
4%
50
150 200 250
105
104
P2
104
103
VPD450
J
105
105
104
103
102
103
150
100
50
102
100
(x 1,000)
E
Day 3 10 µM VPD450
C
20.6%
50
150 200 250
0
Count
100
7-AAD
G
P2
0 10 2030405060 70 8090
105
104
20.9%
102
BrdU APC
P2
50
D
Day 3 1 µM VPD450
B
104
Day 3 No-load Cells
A
102
103
104
105
102
103
104
105
VPD450
Figure 2. Human PBMCs were loaded with varying concentrations of VPD450 dye or left untreated
as controls (no-load cells), then stimulated for five days with PHA. Cell aliquots were pulsed for 1
hour with BrdU, harvested, then stained with APC Anti-BrdU antibody (APC BrdU) and 7-AAD at
days 3 (A–F) and 5 (G–L). The top panels (A–C and G–I) illustrate BrdU and 7-AAD staining, while
the lower panels (D–F and J–L) illustrate the corresponding VPD450 histograms. Labeling cells with
VPD450 at higher concentrations negatively affects the ability of the cells to proliferate at day 3.
There was a reduction in the percentage of BrdU+ cells in cells prelabeled with 10 μM of VPD450 (C)
compared to both the control cell (no-load cells) (A) and the 1-μM VPD450-loaded cell populations
(B). On day 5 the phenomena were reversed. The no-load cells and the 1-μM VPD450-loaded cell
population had much lower percentages of BrdU+ cells (G and H) compared to the 10-μM VPD450loaded cell population (I). The 10-μM VPD450 load affected the ability of cells to proliferate in
response to PHA when compared to the control cells and the 1-μM VPD450-loaded cell population.
BD Biosciences
Technical Bulletin
January 2012
Technique for Loading Cells with BD Horizon™ Violet Proliferation Dye 450 (VPD450)
Page 5
This same phenomenon was observed in the mouse system as shown by the
following data for day 2 responses of cells stimulated with anti-CD3ε/anti-CD28
antibodies.
(x 1,000)
(x 1,000)
105
10 µM VPD450
P3
0.3%
103
50
100 150 200 250
D
102
103
102
50
100 150 200 250
39.8%
102
103
104
103
50
P3
104
39.5%
1 µM VPD450
104
P3
104
40.7%
C
105
DMSO Control
B
105
105
No-load Cells
102
BrdU APC
A
50
100 150 200 250
(x 1,000)
100 150 200 250
(x 1,000)
7-AAD
104
105
102
103
104
105
10 20 30 40 50 60
0
0 10 20 30 40 50 60 70 80
75
50
25
103
0
102
H
G
100
100
75
50
0
Count
F
25
E
102
103
104
105
102
103
104
105
VPD450
Figure 3. C57BL/6 mouse splenocytes were either loaded with varying concentrations of
VPD450, dimethylsulfoxide (DMSO) as an excipient control, or left as untreated controls, and then
stimulated with CD3ε and CD28 antibodies for two days. Cells were loaded with BrdU prior to
harvest, and then stained for incorporating BrdU (APC BrdU) and 7-AAD. The top panels (A–D)
illustrate BrdU and 7-AAD staining. The bottom panels (E–H) illustrate the corresponding VPD450
histograms. The control cells (no-load cells) (A) and the 1-μM VPD450-loaded cell population (C)
demonstrated similar percentages of BrdU+ cells (40.7% and 39.5%, respectively). The 10-μM
VPD450-loaded cell population (D) was negatively affected by the dye in its ability to incorporate
BrdU (0.3% BrdU+ cells). To confirm that the decrease in proliferation was not due to DMSO
(which was used as a solvent for VPD450), a DMSO control was included (B). A similar percentage
of DMSO-treated cells incorporated BrdU when compared to the no-load cells control group and
the 1-μM VPD450-loaded cell populations, suggesting that the concentration of DMSO used as
solvent for VPD450 was not significantly toxic to the cells.
BD Biosciences
Technical Bulletin
January 2012
Technique for Loading Cells with BD Horizon™ Violet Proliferation Dye 450 (VPD450)
We observed that labeling cells with 10 μM of VPD450 led to a significant
decrease in cell proliferation. Therefore, we tested whether the dye also caused
apoptosis by assaying for the apoptotic marker, cleaved poly (ADP-ribose)
polymerase (PARP) (Figure 4). This data suggests that VPD450 does not lead to
significant apoptosis in PBMCs, but may lead to an increase in apoptosis in mouse
splenocyte populations when higher concentrations of dye are used.
103 104 105
4%
102
102
103
104
102
105
PBMC + 10 µM VPD450
C
4%
102
103 104 105
4%
102
Cleaved PARP PE
PBMC + 1 µM VPD450
B
103 104 105
PBMC No-load
A
103
104
102
105
103
104
105
VPD450
Spleen No-load
Spleen + 1 µM VPD450
103
104
105
103
102
102
102
14%
104
104
1.7%
103
103
104
1.3%
Spleen + 10 µM VPD450
F
105
105
105
E
102
Cleaved PARP PE
D
102
103
104
105
102
103
104
105
VPD450
Figure 4. PBMCs or BALB/c mouse spleen cells were loaded for 10 minutes with either 1 μM of
VPD450, 10 μM of VPD450, or left untreated as controls (no-load). PBMCs were incubated for
three days with anti-CD3ε antibody whereas mouse splenocytes were stimulated for two days
with anti-CD3ε + anti-CD28 antibodies. The cells were harvested and then stained with PE-AntiCleaved PARP (Cleaved PARP PE) (blue events). The PBMCs do not appear to have any increased
frequency of apoptosis compared to the control cells (A–C). Mouse spleen cells appear to be more
sensitive to the dye. The 10-μM concentration of VPD450 (F) led to an increase in the percentage
of apoptotic cells compared to spleen no-load cells (D) and cells treated with 1 μM of VPD450 (E).
BD Biosciences
Technical Bulletin
January 2012
Technique for Loading Cells with BD Horizon™ Violet Proliferation Dye 450 (VPD450)
Page 7
Cell loading
Another important detail that we chose to address was the technique of loading
cells with VPD450. Loading cells with VPD450 would appear to be a simple
process. However, since the dye binds to plastic, we found that it is important
to add the dye directly to the diluted cell suspension rather than adding the dye
to an empty tube first and then transferring the cells to that tube. The following
experiment underscores the importance of adding the dye directly to the diluted
cells.
38.6%
50
100
150
105
48.3%
100
150
200 250
(x 1,000)
0.6%
P2
103
103
50
200 250
(x 1,000)
P2
102
102
102
103
103
P2
10 µM VPD450 added
directly to diluted cells
D
104
104
37.6%
10 µM VPD450 diluted in
PBS then added to cells
C
105
105
105
104
P2
102
BrdU APC
1 µM VPD450 added
directly to diluted cells
B
104
1 µM VPD450 diluted in
PBS then added to cells
A
50
100
150
200 250
(x 1,000)
50
100
150
200 250
(x 1,000)
H
50
100
100
150
150
100
102
103
104
105
0
0
50
50
0
0
Count
50 100 150 200 250
G
200
F
150
7-AAD
E
102
103
104
105
102
103
104
105
102
103
104
105
VPD450
Figure 5. Mouse splenocytes were loaded for 10 minutes at 37°C with VPD450 using two
different loading techniques—either adding the dye directly to diluted cells (B, D, F, and H) or
diluting the dye in PBS and then transferring the solution to a dispersed cell pellet (A, C, E, and
G). The stock concentration of VPD450 is 1 mM in DMSO. To load cells with 1 μM of VPD450, we
added 1 μL of VPD450 to 1 mL of cell suspension in 1X PBS. Alternatively, we diluted the VPD450
in 1X PBS first and then transferred the solution to a dispersed cell pellet. A similar protocol was
followed for the higher concentration 10-μM VPD450 load. The cell concentration used in this
experiment was 1 x 107 cells/mL. When comparing the two different dye loading techniques
in 1-μM loads, the group with the dye added to the resuspended cells in the 1-mL volume (B)
showed discernable VPD450 peak generations whereas the 1-μM volume diluted in PBS prior
to adding to cells (A) did not show any defined peaks. Alternatively, the 10-μM VPD450 load,
when added to cells directly, severely inhibited the cells’ responses and allowed for minimal BrdU
incorporation as expected (D). However, when the 10-μM dose of VPD450 dye was first diluted
in PBS and then transferred to a cell population (C), the percentage of BrdU incorporation did not
decrease, and the VPD450 peaks were very well defined as seen in (G). We hypothesize that as the
dye was diluted in PBS prior to addition to the cells, some VPD450 dye bound to the tube thereby
lowering its concentration. The remaining dye was sufficient to label the cells and was then at a
low enough concentration so that it did not interfere with cell proliferation, thereby leading to
well-defined peaks as seen in (G). Based on these observations, we recommend that the DMSOdiluted dye be added directly to the cell suspension. Because the dye binds to plastic, we do not
recommend diluting the dye in PBS in a separate tube prior to adding it to the cells.
BD Biosciences
Technical Bulletin
January 2012
Technique for Loading Cells with BD Horizon™ Violet Proliferation Dye 450 (VPD450)
Dye effects on the cell population
Optimal cell concentrations and VPD450 dye load concentrations are critical to
having a successful experiment. Therefore, we also chose to examine the potential
effects that this dye can have on the ability of a cell population to produce
cytokines or to transduce signals in response to exogenously added recombinant
cytokines. Two techniques—intracellular cytokine (IC) staining and
BD™ Cytometric Bead Array (CBA)—were used to evaluate cytokine production.
IC staining determines the percentage of cells within a given population
expressing a given cytokine while CBA analysis determines the amount of
secreted cytokine produced by a population of cells.
Q4
102
103
104
Q3
103
104
105
103
104
105
CBA SN mTNF
Spleen1 µM VPD450
10 µM VPD450
105
104
103
105
104
2.3%
103
104
Q4
Q3
102
105
103
104
105
Q3-1
102
103
Q4-1
104
105
Q4-1
Q3-1
102
103
104
4 hr
18 hr
PBMC1 µM VPD450
10 µM VPD450
291.5 pg/mL
99.8 pg/mL
7.1 pg/mL
3,586.7 pg/mL
1,393.7 pg/mL
91.6 pg/mL
4B
9.8%
PBMC + 1 µM VPD450
Q1
Q2
9.5%
6%
104
105
105
6.7%
Q2
Q4
Q3
102
103
104
105
4C
105
PBMC No-load
Q1
104
19.4%
103
4.0%
CBA SN hIL-2
4A
Q2-1
CD4 PerCP-Cy5.5
3D
Q2
0.3%
104
10 µM VPD450
Q1-1
102
Q4-1
102
Q3-1
Q4
Q3
102
105
103
28.4%
103
9.0%
3C
Q2-1
105
105
26.3%
Q1-1
TNF APC
1 µM VPD450
3B
Q2-1
104
Spleen No-load
102
8.9%
Q1
PBMC + 10 µM VPD450
Q1
Q2
6.2%
9.5%
103
1,977 pg/mL
2,118 pg/mL
189 pg/mL
7.8%
104
PBMC + 10 µM VPD450
2C
Q4
Q3
102
103
104
105
102
18 hr
54 pg/mL
79 pg/mL
11 pg/mL
Q1-1
103
105
4 hr
Spleen1 µM VPD450
10 µM VPD450
102
104
2D
CBA SN mIL-2
103
102
TNF APC
105
3A
0.8%
Q2
CD4 FITC
104
1D
Q4
Q3
102
CD4 PerCP-Cy5.5
PBMC + 1 µM VPD450
Q1
103
104
105
Q4
102
15.2%
103
IL-2 PE
105
105
2B
Q2
1.5%
102
Q3
103 104
105
105
2.2%
Q1
103
104
PBMC No-load
2A
Q2
102
103
8.8%
10 µM VPD450
Q1
102
Q4
102
1C
Q2
102
Q3
1 µM VPD450
Q1
103 104
8.2%
102
IL-2 PE
102
1B
Q2
102
Spleen No-load
Q1
103 104
105
1A
Q4
Q3
102
103
104
105
CD4 FITC
4 hr
77 pg/mL
176 pg/mL
90 pg/mL
18 hr
389 pg/mL
552 pg/mL
211 pg/mL
4D
CBA SN hTNF
4 hr
PBMC1 µM VPD450
10 µM VPD450
200.5 pg/mL
167.9 pg/mL
111.8 pg/mL
18 hr
795.6 pg/mL
839.8 pg/mL
179.0 pg/mv
Figure 6. IC staining and CBA analysis of mouse splenocytes and human PBMCs stimulated with PMA + ionomycin ± BD GolgiStop™ (monensin)
protein transport inhibitor and cultured for either 4 hours or overnight. Mouse splenocytes or human PBMCs were isolated, loaded with either 1
μM of VPD450, 10 μM of VPD450, or left as controls (no-load). Cells were washed, then cultured at 2 x 106 cells/mL in complete medium with PMA
(50 ng/mL) and ionomycin (1 μg/mL) ± BD GolgiStop (monensin) for either 4 hours (for intracellular cytokine staining and CBA analysis) or cultured
overnight (for CBA analysis). For IC staining, cells were harvested, fixed, and permeabilized with BD Cytofix/Cytoperm™ fixation/permeabilization
solution, then stained with fluorescent anti-cytokine antibodies: either IL-2 PE, TNF APC, and CD4 PerCP-Cy™5.5 for mouse cells or IL-2 PE or TNF
APC and CD4 FITC for PBMCs. For the CBA assay, cells were set up as above but without BD GolgiStop and the culture supernatants were harvested
at 4 hours and 18 hours, then analyzed with the BD CBA Mouse or Human Th1/Th2 Cytokine Kit. Panels 1A–C are mouse splenocytes stained
with IL-2 PE vs CD4 PerCP-Cy5.5, panels 2A–C are human PBMCs stained with IL-2 PE vs CD4 FITC, panels 3A–C are mouse splenocytes stained
with TNF APC vs CD4 PerCP-Cy5.5, and panels 4A–C are human PBMCs stained with TNF PE vs CD4 FITC. The corresponding CBA analysis data is
found in Figures 1D through 4D. This data demonstrates that there was a decrease in the ability of cells to produce IL-2 when loaded with 10 μM
of VPD450 in both the mouse and the human system. Figures 1C and 2C were compared with the no-load figures 1A and 2A. The human system
demonstrated a decrease in IL-2 expression in the 1-μM VPD450 load (2B) when compared to the no-load cells (2A). The mouse system did not show
any decrease in IL-2 production at the 1-μM VPD450 load. The CBA data confirms the observations seen with the IC staining as shown in panels 1D
and 2D. TNF production does not follow the same trend as IL-2. The decrease in TNF production by activated cells labeled with 10 µM of VPD450
was much less than IL-2 production. Comparing panels 3A and 3B, the percentage of cells positive for TNF was similar, while there was a reduction
in the percentage of positive cells for the 10-μM VPD450 condition (3C). In the human system, there appeared to be no significant difference in
the generation of TNF-positive cells as shown in panels 4A–4C. However, the level of soluble TNF produced by cells labeled with 10 µM of VPD450
was signicantly less when compared with the control or the 1-µM VPD450-labeled group. The results suggest that VPD450, when loaded at higher
concentrations, can impact a cell’s ability to produce specific cytokines.
BD Biosciences
Technical Bulletin
January 2012
Technique for Loading Cells with BD Horizon™ Violet Proliferation Dye 450 (VPD450)
Page 9
Effects of dye on cell signaling events
As previously observed, we know that the VPD450 dye at high concentrations
decreases the ability of cells to secrete certain cytokines—especially IL-2. We
chose to evaluate the cell’s ability to respond to IL-2 after VPD450 labeling and
compared to a no-load control. When cells are stimulated with IL-2, they signal
by phosphorylating Stat-5 (pY694). Human PBMCs and mouse splenocytes were
isolated, then loaded with either 10 μM of VPD450, 1 μM of VPD450, or left
as no-load controls. Cells were plated at 2.5 x 106 cells/well (2 mL) in a 12-well
tissue culture plate, then incubated for 30 minutes at 37ºC (prior to the addition
of 300 ng of recombinant human IL-2) or were left untreated. Cells were
incubated for 15 minutes, then fixed using BD Cytofix™ fixation buffer, followed
by permeabilization with BD Phosflow™ Perm Buffer III prior to staining with
anti-Stat5 (pY694) Alexa Fluor® 647 and anti-CD4 FITC. In the human system,
the VPD450 dye at either concentration had no effect on the loaded cell
population’s ability to phosphorylate Stat5 (pY694) when compared to the
no-load controls. However, mouse splenocytes were affected by the 10-μM
VPD450 dye concentration as seen by the nearly 50% decrease in the percentage
of Stat5 (pY694)–positive cells.
A
PBMC No-load
60
40
30
2
10
D
3
10
10
4
0
5
10
Spleen No-load
2
3
10
10
10
4
0
5
10
+1 µM VPD450
E
40
Count
Count
15.9%
30
20
10
102
103
104
105
0
102
103
104
102
105
40
35
30
25
20
15
10
5
0
103
104
105
+10 µM VPD450
F
50
16.5%
30
10
10
0
40
20
20
10
29.7%
50
Count
Count
Count
30
60
31.2%
50
40
+10 µM VPD450
70
60
20
Count
C
70
32.4%
50
40
35
30
25
20
15
10
5
0
+1 µM VPD450
B
7.7%
102
103
104
105
Stat-5 (pY694) Alexa Fluor® 647
Figure 7. PBMCs (A–C) or BALB/c mouse splenocytes (spleen) (panels D–F) were loaded with
varying concentrations of VPD450 or left as untreated controls (no-load). Cells were plated at
2.5 x 106 cells/well (2 mL) in a 12-well tissue culture plate and incubated for 30 minutes at 37ºC
(prior to the addition of 300 ng of recombinant human IL-2) or were left untreated. The cells were
incubated for 15 minutes, then fixed using BD Cytofix fixation buffer, followed by permeabilization
with BD Phosflow Perm Buffer III prior to staining with anti-Stat5 (pY694) Alexa Fluor® 647 and
anti-CD4 FITC. The black line shows the unstimulated cells and the colored lines show the cells
stimulated with IL-2. PBMCs loaded with VPD450 did not appear to show a significant difference
in their ability to signal in response to IL-2 (A–C). Mouse spleen cells (gated on CD4+ cells) showed
a 50% decrease in their ability to signal in response to IL-2 when loaded with 10 μM of VPD450
(F) compared to the no-load cells (D) and cells prelabeled with 1 μM of VPD450 (E).
BD Biosciences
Technical Bulletin
January 2012
Technique for Loading Cells with BD Horizon™ Violet Proliferation Dye 450 (VPD450)
Controls
We have demonstrated that the optimal concentration of VPD450 is 1 μM and
the optimal cell number is greater than or equal to 2 x 106 cells in 1 mL. Next
we determined the controls necessary for using VPD450. We observed that a
VPD450-loaded day 0 control is important when setting up an experiment to
account for the initial load characteristics of each cell population. The initial cell
load might not be a single distinct peak, but might appear as two distinct peaks
as seen in panel A of Figure 8.
103
104
105
Day 3
D
25
50
50
100
75
150
100 125
200
50 100 150 200 250 300 350
102
Day 2
C
0
0
50 100 150 200 250 300
0
Count
Day 1
B
0
Day 0
A
102
103
104
105
102
103
104
105
102
103
104
105
VPD450
Figure 8. Mouse splenocytes were enriched for CD4+ cells using a positive panning selection
technique. The enriched CD4+ cells were loaded with 1 μM of VPD450, then stimulated with antiCD3ε + CD28 (2 µg/mL) antibodies for three days. Cells were harvested at each day of culture
including an aliquot for a day 0 time point to determine how the cell population loaded with the
VPD450 dye. Panel A = day 0 load, Panel B = day 1 time point, Panel C = day 2 time point, and
Panel D = day 3 time point. The cell population did not load as a single peak at day 0 (A). If this
control was not present, it would be assumed that the cells had completed one round of division
at day 1 (B). At day 2 (C), the cell population started to divide, which is evident by the three
additional peaks, and at day 3 (D), the cell population had completed five rounds of division.
The day 0 load is very important for recording the baseline VPD450 load.
Conclusions
VPD450 can be very useful in determining the kinetics of when key events are
occurring in an experimental system. The critical parameters for establishing a
successful experiment are cell number, dye concentration, and load technique.
Whenever a dye is added to cells, it will have some effect on the population’s
ability to respond to stimuli. The key is to use as little dye as possible which
should minimize unwanted effects of the dye in your experimental system. It is
extremely important to include a no-dye load control for comparison in your
experimental setup since this control will provide the optimal response of the
cell population to each treatment condition. An additional zero timepoint dyeload control is also recommended. This is useful for determining how well the
cell population loaded with dye initially. Cell populations do not necessarily
display as one defined peak—they may show a double peaks of VPD450
fluorescence intensities.
From experiments we determined that loading higher numbers of cells (1 x 107
to 2 x 107 cells/mL) with the 1-µM dose of VPD450 dye are recommended when
compared to loading low cell numbers (1 x 106 cells) with the same dose based
on the decrease in BrdU incorporation observed compared to the no-load cell
controls. We have also shown that using high dye concentrations of 10 μM
negatively affects the ability of cells to proliferate and to secrete certain cytokines.
The high concentration of VPD450 also led to decreased Stat5 (pY694) signaling
by mouse cells that were stimulated with IL-2. Based upon our experiments, it
has been determined that the optimal concentration for using this dye is 1 μM
and the optimal cell loading concentration is between 1 x 107 to 2 x 107 cells/mL.
BD Biosciences
Technical Bulletin
January 2012
Technique for Loading Cells with BD Horizon™ Violet Proliferation Dye 450 (VPD450)
Page 11
References
1. Ruitenberg JJ, Boyce C, Hingorani R, Putnam A, Ghanekar SA. Rapid assessment of in vitro
expanded human regulatory T cell function. J Immunol Methods. 2011;372:95-106.
2. L yons AB. Analysing cell division in vivo and in vitro using flow cytometric measurement of CFSE
dye dilution. J Immunol Methods. 2000;243:147-154.
3. Lyons AB, Doherty KV. Flow cytometric analysis of cell division by dye dilution. Curr Protoc
Cytom. 2004;9:1-9.
4. Parish CR. Fluorescent dyes for lymphocyte migration and proliferation studies. Immunol Cell
Biol. 1999;77:499-508.
BD Biosciences
Technical Bulletin
January 2012
Technique for Loading Cells with BD Horizon™ Violet Proliferation Dye 450 (VPD450)
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
Ficoll-Paque™ is a trademark of GE Healthcare.
Cy™ is a trademark of Amersham Biosciences Corp. Cy dyes are subject to proprietary rights of Amersham
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