1. INTENDED USE
BD™ stabilizing fixative (3X concentrate)
is intended for preserving
immunofluorescence staining of human
peripheral blood cells with monoclonal
antibodies prior to flow cytometric
analysis.
BD™ Stabilizing Fixative
3X concentrate—Catalog No. 339860
2. SUMMARY AND EXPLANATION
04/2015
Fluorochromes requiring efficient energy
transfer to Cy™7* (PE-Cy7 and
APC-Cy7†) and H7 (APC-H7), can break
down and begin to show emission from
the donor part of the fluorochrome. This
breakdown can be caused by exposure to
light during the staining process, and by
prolonged storage in formaldehydecontaining buffers after staining.
BD stabilizing fixative decreases the rate
of this second type of breakdown in these
tandem conjugates.
23-7725-05
IVD
BD, BD Logo and all other trademarks are property of
Becton, Dickinson and Company. © 2015 BD
3. PRINCIPLES OF THE PROCEDURE
Becton, Dickinson and Company
BD Biosciences
2350 Qume Drive
San Jose, CA 95131 USA
When using a lyse/wash (LW) staining
procedure for Cy7 and H7 tandem
conjugates, resuspend the washed cells in
1X BD stabilizing fixative rather than 1%
paraformaldehyde (PFA) solution.
Benex Limited
Pottery Road, Dun Laoghaire,
Co. Dublin, Ireland
Tel +353.1.202.5222
Fax +353.1.202.5388
BD Biosciences
European Customer Support
Tel +32.2.400.98.95
Fax +32.2.401.70.94
[email protected]
Becton Dickinson Pty Ltd,
4 Research Park Drive,
Macquarie University Research Park,
North Ryde NSW 2113, Australia
* Cy™ is a trademark of GE Healthcare. This
product is subject to proprietary rights of GE
Healthcare and Carnegie Mellon University, and is
made and sold under license from GE Healthcare.
This product is licensed for sale only for in vitro
diagnostics. It is not licensed for any other use. If
you require any additional license to use this
product and do not have one, return this
material, unopened, to BD Biosciences, 2350
Qume Drive, San Jose, CA 95131, and any money
paid for the material will be refunded.
† APC-Cy7: US patent 5,714,386
Becton Dickinson Limited,
8 Pacific Rise, Mt. Wellington,
Auckland, New Zealand
bdbiosciences.com
[email protected]
1
regulations. Never pipette by mouth.
Wear suitable protective clothing,
eyewear, and gloves.
4. REAGENT
Reagent provided
BD stabilizing fixative (3X concentrate) is
provided as 90 mL of a proprietary
buffered solution containing less than 3%
formaldehyde. This quantity is sufficient
for 500 tests when used in BD LW
procedures.
Dilution instructions
Dilute 1 part of the 3X concentrate with
2 parts of room temperature (20°C–
25°C) deionized (DI) water. The prepared
1X solution is stable for one month when
stored in a polystyrene container at 2°C–
8°C.
BD stabilizing fixative is delivered as a 3X
concentrate for use on the BD FACS™
Lyse Wash Assistant (LWA).
WARNING Do not store 1X
BD stabilizing fixative in high-density
polyethylene (HDPE) bottles because
performance can be affected.
Reagent contains 2.81% formaldehyde,
CAS number 50-00-0, and 4.01%
disodium tetraborate decahydrate, CAS
number 1303-96-4.
Storage and handling
BD stabilizing fixative (3X) is stable until
the expiration date shown on the bottle
label when stored as directed.
Danger
H341 Suspected of causing genetic
defects.
H350 May cause cancer. Route of
exposure: Inhalative.
H360 May damage fertility or the
unborn child.
Do not use the reagent if you observe any
change in appearance. Precipitation or
discoloration indicates instability or
deterioration.
H317 May cause an allergic skin
reaction.
WARNING Do not store 3X
BD stabilizing fixative in LWA tanks for
more than five months. Although LWA
tanks are made of HDPE, BD stabilizing
fixative can be stored for up to five
months if the tanks are pre-washed with
DI water.
Wear protective gloves / eye protection.
Wear protective clothing. IF ON SKIN
(or hair): Remove/Take off immediately
all contaminated clothing. Rinse skin
with water/shower. IF exposed or
concerned: Get medical advice/attention.
If skin irritation or rash occurs: Get
medical advice/attention. Dispose of
contents/container in accordance with
local/regional/national/international
regulations.
WARNING Do not store samples stained
with AmCyan conjugates in BD stabilizing
fixative. Exposure of these conjugates to
BD stabilizing fixative can cause increased
spillover into the FITC channel.
Precautions
For In Vitro Diagnostic Use.
WARNING Do not store samples stained
with PE conjugates in BD stabilizing
fixative because this can cause a decrease
in staining intensity.
WARNING All biological specimens and
materials coming in contact with them are
considered biohazards. Handle as if
capable of transmitting infection1,2 and
dispose of with proper precautions in
accordance with federal, state, and local
2
•
5. INSTRUMENT
Samples fixed with BD stabilizing fixative
can be analyzed by flow cytometers
equipped with appropriate computer
hardware and software. The flow
cytometer must be equipped to detect light
scatter and the appropriate fluorescence,
and be equipped with appropriate analysis
software (such as BD CellQuest™,
BD CellQuest™ Pro, BD FACSDiva™, or
BD FACSCanto™ clinical software) for
data acquisition and analysis. See the
cytometer user’s guide for instructions.
Preparing samples
Stain whole blood specimens following
the instructions in the appropriate reagent
IFU. Lyse red blood cells as directed using
diluted (1X) BD FACS lysing solution. Use
care to protect the tubes from direct light.
Perform the procedure at room
temperature (20°C–25°C).
1. Add the appropriate volume of
fluorochrome-conjugated monoclonal
antibody to 100 µL of whole blood in
a 12 x 75-mm polystyrene test tube.
See the antibody vial label for volume.
6. PROCEDURE
Reagent provided
BD stabilizing fixative, 3X concentrate
(Catalog No. 339860)
2. Vortex gently and incubate for 15 to
30 minutes in the dark at room
temperature (20°C–25°C).
Reagents and materials required but not
provided
•
1X BD FACS™ lysing solution. For
dilution instructions and warnings, see
the product instructions for use (IFU).
•
Falcon®‡ disposable 12 x 75-mm
polystyrene test tubes or equivalent
•
BD Biosciences monoclonal antibodies
to human leucocyte antigens
•
Vortex mixer
•
Centrifuge
•
Micropipettor with tips
•
BD CellWASH™ (Catalog No. 349524)
or a wash buffer of phosphate buffered
saline (PBS) with 0.1% sodium azide
•
Stain buffer solution consisting of PBS
plus 0.2% bovine serum albumin (BSA)
with 0.1% sodium azide
1X BD stabilizing fixative. See Dilution
instructions on page 2.
3. Add 2 mL of 1X BD FACS lysing
solution to each tube.
4. Vortex the tubes gently and incubate
for 10 minutes in the dark at room
temperature.
5. Centrifuge the tubes at 300g for
5 minutes.
6. Aspirate the supernatant.
7. Add 2 mL of BD CellWASH solution
or stain buffer to each tube. See the
fluorochrome-conjugated monoclonal
antibody IFU for more information.
8. Centrifuge the tubes at 300g for
5 minutes.
9. Aspirate the supernatant.
10. Add 0.5 mL of 1X BD stabilizing
fixative to the tubes.
CAUTION Do not use BD stabilizing
fixative for multicolor analysis using a
PE or AmCyan conjugate. See the
‡ Falcon is a registered trademark of Corning
Incorporated.
3
fluorochrome-conjugated monoclonal
antibody IFU for more information.
Table 2 Percent spillover shown at 2 hours,
24 hours, and 48 hours using PBS, 1% PFA, or
BD stabilizing fixative on CD20 APC-Cy7
11. Vortex the tubes gently and incubate
for 30 minutes in the dark at 2°C–
8°C.
12. Mix the tubes thoroughly before
analysis. Store the tubes at 2°C–8°C in
the dark until analyzed. We
recommend analyzing within 24 hours
of staining.
Buffer
CAUTION Some PE-Cy7, APC-Cy7, and
APC-H7 conjugates show changes in their
emission spectra with prolonged exposure
to paraformaldehyde. For overnight
storage of stained cells, wash and
resuspend in buffer without
paraformaldehyde after 1 hour of
fixation.
Change
in %
spillover
%
%
%
between
spillover spillover spillover 48 h and
2h
24 h
48 h
2h
PBS
25.02
23.67
26.86
1.84
PFA
23.97
27.09
99.72
75.75
BD stabilizing
fixative
24.06
28.73
34.92
10.86
Table 3 Percent spillover shown at 2 hours,
24 hours, and 48 hours using PBS, 1% PFA, or
BD stabilizing fixative on CD8 APC-Cy7
Buffer
Change
in %
spillover
%
%
%
between
spillover spillover spillover 48 h and
2h
24 h
48 h
2h
7. RESULTS
PBS
17.93
16.60
18.30
0.37
The following representative data were
obtained with peripheral blood samples
treated as instructed in this IFU. Final resuspension was in PBS, PBS with 1%
paraformaldehyde, or BD stabilizing
fixative.
PFA
18.13
19.87
38.65
20.52
BD stabilizing
fixative
17.80
23.22
27.45
9.65
NOTE
Storing stained cells in
BD stabilizing fixative can result in a
slight degradation of forward scatter
performance.
Table 1 Percent spillover shown at 2 hours,
24 hours, and 48 hours using PBS, 1% PFA, or
BD stabilizing fixative on CD45 APC-Cy7
Buffer
8. LIMITATIONS
Change
in %
spillover
%
%
%
between
spillover spillover spillover 48 h and
2h
24 h
48 h
2h
PBS
17.26
PFA
16.95
BD stabilizing
fixative
17.46
16.23
18.26
1.00
18.47
44.73
27.78
21.17
25.67
8.21
•
BD stabilizing fixative is specifically
formulated for use with BD FACS™
brand flow cytometers.
•
EDTA is the anticoagulant of choice. BD
has limited information concerning use
of other anticoagulants, such as heparin.
WARRANTY
Unless otherwise indicated in any applicable BD
general conditions of sale for non-US customers,
the following warranty applies to the purchase
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of these products.
THE PRODUCTS SOLD HEREUNDER ARE WARRANTED ONLY TO
CONFORM TO THE QUANTITY AND CONTENTS STATED ON THE LABEL
OR IN THE PRODUCT LABELING AT THE TIME OF DELIVERY TO THE
CUSTOMER. BD DISCLAIMS HEREBY ALL OTHER WARRANTIES,
EXPRESSED OR IMPLIED, INCLUDING WARRANTIES OF
MERCHANTABILITY AND FITNESS FOR ANY PARTICULAR PURPOSE AND
NONINFRINGEMENT. BD’S SOLE LIABILITY IS LIMITED TO EITHER
REPLACEMENT OF THE PRODUCTS OR REFUND OF THE PURCHASE
PRICE. BD IS NOT LIABLE FOR PROPERTY DAMAGE OR ANY
INCIDENTAL OR CONSEQUENTIAL DAMAGES, INCLUDING PERSONAL
INJURY, OR ECONOMIC LOSS, CAUSED BY THE PRODUCT.
REFERENCES
1. Protection of Laboratory Workers from
Occupationally Acquired Infections; Approved
Guideline–Third Edition. Wayne, PA: Clinical and
Laboratory Standards Institute; 2005. CLSI
document M29-A3.
2. Centers for Disease Control. Perspectives in disease
prevention and health promotion update: universal
precautions for prevention of transmission of human
immunodeficiency virus, hepatitis B virus, and other
bloodborne pathogens in health-care settings.
MMWR. 1988;37:377-388.
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