*2.4.1 Nuclear staining and mounting*

*Erythrocyte - A Peripheral Biomarker for Infection and Inflammation*

1 RPMI 1640 11875 Thermo

2 Foetal Calf Serum A3160401 Thermo

3 Trypsin 9035-81-8 Sigma-

6 Paraformaldehyde 30525–89-4 Sigma-

**number**

Fisher Scientific

Fisher Scientific

Aldrich

Fisher Scientific

Aldrich

Aldrich

10010023 Thermo

34369–07-8 Sigma-

**Company Amount used Quantity** 

5 ml

4% paraformaldehyde in PBS, with 7.4 pH

**T75 flask**

complete media

4 ml

3 ml

100 μl

1 ml

45 ml 15 ml of

**S. N. Chemical Catalogue** 

4 Phosphate-

5 Adenosine

**Table 1.**

buffered saline (PBS)

5- triphosphate disodium salt hydrate (ATP)

*Chemicals used for the tissue culture experiments.*

the cells were returned to RPMI media +10% FCS in both A1 and A2 flasks. Then, 100 μl of 1 mM adenosine triphosphate (ATP) was added to the A1 flask to convert the normal HFAs into reactive HFAs [23]. After 24 hours of incubation at 37°C, in a 5% CO2 humidified incubator, the cells were washed three times with PBS. The cells were subsequently detached from both flasks by adding 4 ml of trypsin and were incubated at 37°C in a humidified incubator, for 3 minutes. After incubation, the cells were transferred into two falcon tubes (15 ml tubes, labelled as A1 and A2) and were centrifuged at 1600 g rpm for 3 minutes. Trypsin was removed from the tubes by washing three times with 3 ml PBS. The cell samples from both tubes were transferred into 2 ml Eppendorf tubes and were microfuged for 1 minute, to remove PBS. Subsequently, the Eppendorf tubes containing cell pellets were snap frozen in liquid nitrogen and stored at -80°C for the proteomics experiment, using SP3 protocol.

Six poly-L-lysine coated coverslips, three labelled as A1 and three as A2 were placed in a six well plate, containing 2 ml of fresh RPMI media with 10% FCS. The plates were incubated at 37°C, in a 5% CO2 humidified incubator to grow until

The cells in three of the six wells, were treated with 1 mM ATP to convert them into reactive (A1) astrocytes, whereas the other three coverslips with the HFAs (A2) were not treated with ATP. After 24 hours of incubation all the cells were washed three times with 0.1 M PBS, fixative (4% paraformaldehyde in PBS, 7.4 pH) was then added for 30 minutes, at room temperature. After fixation, both types of HFAs were gently washed three times with PBS and were ready for permeabilization and blocking of the unspecific binding of the proteins. To improve the permeabilization of the antibodies, the HFAs were incubated with 100 μl of 0.1% Triton X 100, for 30 minutes, at 37°C, in a 5% CO2 humidified incubator. The cells were gently washed three times with PBS, before being treated with the blocking solution (100 μl of 5% goat serum) and then the cells were incubated for 30 minutes.

**2.4 Immunocytochemistry of HFAs using anti-GFAP stain**

~70% confluency was achieved.

**58**

The HFAs were rinsed with PBS and were incubated with 1 μg/ml of nuclear stain [4, 6-diamidino-2-phenylindole (DAPI)] for 10 minutes followed by three rinses with PBS. Excess PBS was carefully wiped around the coverslips and the cells were mounted on the glass slides (75 mm x 25 mm and 1 mm thickness), which were clearly labelled with the type of cell and stain. Then a drop of mounting medium, Fluoroshield (ABCAM) was placed in the middle of the slide. The coverslip with the stained cells was carefully lifted using forceps and was placed in an inverted position on top of the slide, air bubbles were removed, and the cells were sealed with a clear nail polish to prevent drying and movement under the microscope. The cells were stored in the dark, at +4°C till image analysis was performed using the NIKON A1 confocal microscope.
