STSM Report
Stephan von Berg, JLU Gießen
The STSM took place at the Roslin Institute to provide a proper training in collecting and
handling porcine macrophages. This is necessary for a cooperative research project
between the Clinic For Swine at the University Gießen and the Roslin Institute. The
research is on the following topic: “Resolving PRRS host genetics based on pig breeds with
different resistance/susceptibility.”
So the goal of the STSM was to obtain the ability to collect and preserve porcine pulmonal
alveolar macrophages (PAM). For this the lung of a freshly euthanized pig is extracted
without lesions if possible. Following the SOP for macrophages isolation in pigs the lung
then is lavaged with PBS three times. Approximately 1 Litre of PBS is needed for the lung
of a 20 kg pig. The recovery rate should be 2/3, the lavage is done under gentle massage
of the pulmonal tissue. Afterwards the lavage fluid is centrifuged at 1500 rpm for 10
minutes. If the remaining pellet contains red blood cells they will be lysed with minimum 5
ml lysis buffer for at least 5 minutes, then the sample must be washed again with PBS and
centrifuged as above. After the lysis another washing is required, again using PBS.
To determine the amount of cells in the sample they need to be counted. For this, a
dilution is made from the pellet which is again re-suspended with 10 ml PBS. This dilution
is 1/100 (10 µl cells + 90 µl PBS → 10 µl + 90 µl trypan blue 0,4%). The coloured dilution
is counted under microscopic vision using an standard counting chamber. The typical cell
count is 2 x 107 to 108 cells per millilitre, only the live cells are counted. Dead cells can be
identified by their blue colour.
Knowing the cells concentration they are brought into the freezing medium (FCS 90% DMSO 10%) with a concentration of usually 5 x 107 cells per millilitre. These 1 ml aliquots
are stored in a freezing box and cooled down carefully at three steps, 24 hours each, first
to -20° C, then -80° C and finally -155° C.
Approximately 3 x 106 Cells are brought into an flask with 5ml RPMI 1640 (10% FBS and
PenStrep) and are stored at 37 °C under 5% CO2 to be checked one day later to asses the
quality of preparation.
Pic. 1
Pic. 2
The above pictures show PAM from the preparation, along with remaining red blood cells
and some cell detritus.
Pic. 3: magnified from Pic. 2
Picture 3 shows 2 macrophages from picture 2, one can see the pseudopodes build by the
cells membrane.
Up to the step of freezing the work will be done at the Clinic For Swine, University Gießen,
the so obtained and preserved PAM samples will then be sent to the Roslin Institute for
further examination and studies. There will also be further pilot experiments to figure out
the best method of storing the samples for transportation.