Richard,
I've done work on a number of RBC antigens, both protein and carbohydrate
epitopes.
Here is our protocol.
RBC STAINING
REFERENCE
Fletcher et al. (1992), Immunology, 75:507
REAGENTS AND MATERIALS
1. Staining Wash - 0.1% BSA/0.1% NaN3 PBS
2. PBS/Azide to be used, where required, in the dilution of antibodies
3. 2. 1% Paraformaldehyde (PFA)
4. Tubes - depending on the centrifuge of choice, the staining may be
performed in 1.5 mL microcentrifuge tubes or 12 X 75 mm polypropylene tubes.
The flow cytometer requires the 12 X 75 mm tubes.
5. PBS .
6. Whole blood in ACD or EDTA anticoagulant.
NOTE
Samples may be used up to 35 days after the day of collection, provided the
controls have been similarly treated.
7. Primary and (where suitable fluorochrome-conjugated antibodies are not
available) secondary fluorochrome-conjugated antibodies.
NOTE
If not already established, antibodies will need to be titrated before
initial use in order to establish the dilution to be used in staining.
Store fluorescein conjugates in foil at 4°C.
8. Controls
The NEGATIVE CONTROL should be either:
a) An irrelevant antibody of the same isotype if using mouse monoclonal
antibodies in the same state, unconjugated or conjugated to the same
fluorochrome, as the labelled antibody to be tested,
OR
Cells known not to express the antigen, e.g. D negative RBC + anti-D. The
POSITIVE CONTROL should contain RBC known to be positive for the antigen to
be tested, i.e. D positive + anti-D.
b) Red cells negative for the antigen of interest if using human serum or
human monoclonal antibodies for the negative control. The positive control
should be cells positive for the antigen of interest (use a heterozygote if
possible).
METHOD
PREPARATION OF RBC
1. Label a tube for each test.
2. Calculate the volume of cell suspension required. Each test requires 2 X
10e6 cells.
3. Fill tubes with PBS. Centrifuge in Immufuge II for 1 min on High. Remove
PBS. Repeat wash.
4. Resuspend at 10e7 cells/mL in staining wash.
STAINING OF RBC
1. Dispense the calculated volume of the cell suspension into each tube
(microcentrifuge tube or 12 X 75 mm polypropylene tube).
2. Centrifuge polypropylene tubes in Immufuge II - 1 min 30 s on High,
microcentrifuge tubes in microcentrifuge for 6 s) and aspirate supernatant.
3. Add 50 µL of the appropriate dilution of the primary antibody.
4. Vortex and incubate at RT for 30 min.
5. Vortex and add Staining Wash.
6. Centrifuge as before and aspirate supernatant.
7. Repeat to give a total of:
2 washes if using 12 X 75 mm tubes
OR
3 washes if using microcentrifuge tubes.
8. If using a DIRECT PROCEDURE (using conjugated primary antibody), add 1.0
mL of PFA, vortex and store, protected from light, at 4oC for analysis
within 24 h.
9. If using an INDIRECT PROCEDURE (using unconjugated primary antibody):
a) Add 20 µL of the appropriate dilution of secondary antibody to each tube.
b) Vortex and incubate at RT for 30 min and protect from light.
c) Vortex on high and add Staining Wash to the level of the collar of the
Immufuge II centrifuge head.
d) Centrifuge as before and aspirate supernatant.
e) Add 1.0 mL of PFA and vortex. Transfer from microcentrifuge tubes to
polypropylene tubes. Store samples protected from light, at 4°C for analysis
within 24 h.
QUALITY CONTROL
At least one Negative and one Positive control should be run with each set
of experiments.
NOTE
If either control, does not give the expected result, ie negative for the
Negative Control and positive for the Positive Control, then the experiment
will have to be repeated.
I have tried to cut out all the QM jargon that infects our methods so I
hope this helps. What I will also add to this is that I prefer to use a
cell negative for the antigen of interest as the negative control and be
careful when using secondary antibodies or IgM antibodies. RBCs love to
agglutinate and this is not good for flow. There are a number of chemical
treatments which have been employed to prevent agglutination but we were
worried that these may change some of the epitopes we were looking at and so
we tried to avoid agglutination by choice of antibody. The best results are
obtained when the primary antibody is not able to cause direct agglutination
(i.e. generally not IgM, but some high avidity IgGs can cause agglutination
too) and the secondary antibody is an Fab' fragment. When first setting up
this method, I did have some problems because certain secondary reagents
were listed as Fab' but were actually F(ab')2 (hopefully this does not
occur any more) leading to agglutination . During the last workshop for
monoclonal antibodies to RBC antigens, we used a very good product from
Jackson ImmunoResearch http://www.jacksonimmuno.com/.
I hope this helps and if you want to ask about particular RBC antigens I may
be able to help too.
Jenny
Jenny Bryant
Flow Cytometry
Australian Red Cross Blood Service - NSW/ACT
153 Clarence St
Sydney, NSW 2000, AUSTRALIA .._|\
Ph: 61 2 9229 4341 / \
FAX: 61 2 9229 4521 \_.-._/<<<<<<
E-mail: jbryant@arcbs.redcross.org.au v
-----Original Message-----
From: Priest, Richard C [mailto:rp15456@GlaxoWellcome.co.uk]
Sent: Monday, 10 January 2000 22:04
To: cyto-inbox
Subject: Red blood cell antigens & staining
Dear Flowers,
I have been asked to look at some red blood cell antigens (non-carbohydrate)
by cytometry but have no experience of doing this.
I am to be given some clinical samples which I will get a once only
look at, therefore things need to be certain before I begin. I would
be interested in any tips that people have to help me on my way eg best
anticoagulant, method of staining etc. I am likely to receive the blood
samples in dribs and drabs and will need to also consider the best way
of processing the samples for meaningful data.
All help will be gratefully received
Thanks
Richard
Richard Priest
Molecular Pharmacology
Glaxo Wellcome R&D Ltd
Gunnels Wood Road
Stevenage
Hertfordshire SG1 2NY
UK
phone: +44 1438 764094
FAX: +44 1438 764818
email: rp15456@glaxowellcome.co.uk
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