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2. disease

SOP | Marion Frankenberger
30-Apr-2011
SOP: Sputum sample processing
Date:
Author: Marion Frankenberger
Experimenter:
Date: 30/04/2011
Experiment description:
Version: 1.0
Please note that sputum is to be processed immediately and without delay.
List of materials
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Sputolysin, Calbiochem #560000 (dilute provided stock solution 1:10 with aqua dest.) alternatively
DDT as a 1% (w/v) monthly stock can be used, dilute 1:10 on the day
Bench Spiromix end-over-end , to hold different size tubes
2 ml Eppendorf tubes, 15 and 50 ml polypropylene tubes
BD Falcon cell strainer 100 µm and 40 µm (#352360 and #352340) or 48 µm gauze
15 ml v-bottom polypropylene tubes #352095 (BD Falcon)
Trypan blue 0.4%, Sigma #T8154
Neubauer counting chamber
Shandon cytocentrifuge
Polylysine microscope slides (# MPR-455-L, Fisher)
Plastic spatula (Brand, 97877 Wertheim,Germany), blue tip pipette
2 ml Sarstedt tubes #72.694.416
1.
2.
3.
4.
5.
Weigh an empty 15 or 50 ml tube
transfer the entire volume of sputum (avoid saliva)°1
reweigh and record the sample weight
based on the weight in g add 2 times the volume in ml of sputolysin (Calbiochem #560000)
disperse sputum by repeated gentle aspiration into a plastic Pasteur pipette, vortex for 15 seconds
and incubate at RT for 15 min on a bench Spiromix°2
6. add PBS, same volume as sputolysin, vortex for 15 sec
7. sieve through cell strainer, (first 100 µm, followed by 40 µm) into a polypropylene tube of
appropriate size, record the volume°3 spin suspension 10 min 800 g at RT.
8. harvest supernatant, transfer it in aliquots of 0.5 - 1 ml into 2ml Sarstedt tubes #72.694.416 and
store them at -20ºC.
9. resuspend cell pellet in 0.5 - 1 ml of PBS °4
10. determine cell number and composition
* Mix 10l of cell suspension with 10l of Trypan blue [dilution factor = 2] or 90l of Trypan blue
[dilution factor = 10]
* Fill a Neubauer chamber and count viable
leukocytes, dead leukocytes and squamous
cells (whether viable or not). (See example in
figure). Cells touching the top and left lines are
counted, cells touching lower and right lines
are not.
Squamous
Spore
Leukocyte
SOP | Marion Frankenberger
30-Apr-11
Calculate the number of viable leukocytes / ml, the percentage of dead leukocytes and the percentage
of squameous cells
a)
number of cells in four 16 field squares (red below) divided by 4. This is multiplied dilution
factor (2 or 10) =
cells x 104 /ml
b)
dead leukocytes x 100
% dead leukocytes
=
[viable + dead leukocytes]
c)
% squamous cells
=
Squamous cells x 100
[Squamous cells + viable leukocytes +
dead leukocytes]
(This is the only calculation involving squamous cells)
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Calculate the total number of cells (cells/ml x total ml)
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Calculate cell count per g sputum = total number of cells / total weight (g) of sputum
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Enter results into the cell number sheet (last page)
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When you have less than 1 x 105 cells stop here and record this as failure and discard the
supernatant
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When you have 1- 2 x 105 cells go ahead and prepare 2 slides as given under 11. and stop here
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When you have > 3 x 105 cells proceed until the end
11.
Prepare 2 slides with about 1 x 105 cells each ( in 100 or 200 µl depending on your centrifuge) in
a cytocentrifuge (18 g = around 400 rpm, for 6 minutes)
12.
Use the rest of the cells for further experiments like mRNA expression or FACS analysis
SOP | Marion Frankenberger
30-Apr-11
Footnotes
°1
picking plugs is done in many laboratories but in our experience gives 10 times less cells
°2
not 37°C incubation since this may lead to ex-vivo release of mediators
°3
gauze can be used for the larger volumes, take care to avoid fluid loss
°4
before centrifugation samples may be too dilute for appropriate counting
Flow cytometry analysis of sputum macrophage subsets
For determination of small and large sputum macrophages before and after RosetteSep isolation the
following monoclonal antibodies according to the manufacturer’s instructions are used: anti-CD66bFITC (#0531, Immunotech, Coulter, Krefeld, Germany), anti-CD16b-PE (#550868, Pharmingen, BD
Sciences, Heidelberg, Germany), anti-CD14-PC5 (#A07765, Coulter, Krefeld, Germany). Small and
large macrophages are determined in percent of all macrophages by first excluding the
CD66b/CD16b-positive granulocytes and further gating on the CD14-positive macrophages. Within
this population small and large macrophages are re-gated in a forward versus side scatter plot to
define their percentage and purity.
Sputum Cell Number Sheet
Donor-Code
Weight of Sputum
Weight of
empty centrifuge
tube (g)
Weight of tube
+ selected sputum
(g)
Weight of
selected sputum (g)
=W
Vol of Sputolysin
added:
(2 x W) (mL)
Volume of PBS added (mL) = Vol of Sputolysin
Haemocytometer Counts (mean of four counted 16 field squares)
Squamous
Leukocytes
%
(viable+dead) Squamous
Viable cells
for
Leukocytes
Non-viable
cells for
Leukocytes
%
Dead
leukocytes
Calculation of Total Number of Cells in Selected Sputum Sample
Weight of
Selected
Sputum
‘W’
Resuspension
volume used
g
Dilution
used
Mean number of
viable cells in
one square 16
field of
haemocytometer
Total viable
cells x 106
in sample
Total Cell
Count per
gram
x 106
ml
x 106/g
Signed:……………………………………………………Date:
Day
Version 1.1 / Apr-11
Month
Year