Handling cells

9.4.1. Splitting cells

Cells were kept at 80-90% confluency by subculturing them every 2-3 days. Old medium was aspirated, cells washed with PBS and detached with trypsin. Trypsin incubation conditions and times varied from cell line to cell line. While HEK293T cells readily detached at room temperature, MEFs needed 2-5 min at 37°C to detach sufficiently.

Trypsin was neutralised by 2-3 volumes of growth medium, cells transferred into clean vials and pelleted at 100xg for 3 min. The supernatant was discarded and cells taken up in 5 ml growth medium. The ratio how cells were split (or cell number) was determined empirically, since different cell lines exhibit different growth rates.

As a rule of thumb for HEK293T cells and most MEF lines, confluent dishes diluted 1:5 acquire their initial confluence at day two after splitting.

For experimental set up, cell number was determined in contrast to sub-culturing.

9.4.2. Counting cells

Cells were counted either using Neubauer chambers or the CasyTT cell-counter.

9.4.2.1. Neubauer chamber

10µl of the cell suspension were pipetted onto the Neubauer chamber, and cells were counted using the microscope. 16 fields from all four quadrants of the chamber

Methods

Handling cells

were counted, and the number of cells multiplied by 10,000 to get the concentration of cells in cells/ml for the suspension.

9.4.2.2. CasyTT

25µl of the cell suspension were diluted in 5 ml filtered CasyTon and measured with the CasyTT using the appropriate program set up once for each cell type. The instrument determines cell number and discriminates duplexes, cells and debris and returns the number of viable cells per millilitre as well as the percentage of viable cells.

9.4.3. FACS Analysis (Fluorescence Activated Cell Sorting)

1x10⁶ cells were seeded into 6cm dishes and infected with CFSE-labelled bacteria the next day for 30-60 minutes as required by experimental set-up and problem posed. The infection was stopped by washing with cold PBS and cells were detached and made single by addition of trypsin as described in “splitting cells”. The cells were taken up in 1ml PBS/10% (F)CS and washed twice with PBS/10%(F)CS.

Samples were kept on ice ant dark until analysis. Exclusively intracellular bacteria were determined by quenching extracellular derived CFSE-signal by addition of trypan blue to a final concentration of 0.2mg/ml (Pils et al., 2006).

9.4.4. Immunostaining for FACS

To measure expression levels of surface proteins, cells were taken into suspension in PBS/10%(F)CS and incubated with antibody against the protein of interest for 30 minutes at 4°C. Cells were washed twice before incubation with fluorescent secondary antibody for another 30 minutes at 4°C. Typically antibody was diluted at 1:200. For 293T-cells, trypsin was diluted 1:5 in PBS to treat surface proteins as carefully as possible.

Samples were measured immediately, but could be fixed in PFA for later analysis as well.

The protocol can also be used for intracellular proteins. In order to make those accessible, cells needed to be fixed in PFA for 20 minutes at room temperature and permeabilised with 0.2% saponin before applying the staining protocol above.

Handling cells

9.4.5. Immunostaining for confocal laser scanning microscopy (CLSM)

Staining of structures had to be done form outside to inside. If intra- as well as extracellular structures had to be stained, extracellular had to be labelled first prior to permeabilisation (0.2% saponin) of the cells to access intracellular structures. All staining steps were conducted using 20µl staining solution (i.e. blocking solution with antibody / biotinylated dye) for 45 minutes in a dark, humid chamber. Preparations were rinsed twice with PBS⁺⁺ after each step. After staining was completed, cover glasses were placed onto mounting medium on a carrier glass slide with the cell side facing the carrier and sealed with nail polish to avoid evaporation of the medium.

Preparations were stored in the dark at 4°C until use.

9.4.6. Gentamicin protection assay

One day before the experiment, 5x10⁵ transfected 293T-cells were seeded into each well of a 24-well plate for infection and 1x10⁶ cells were seeded into 6cm dishes to determine expression of transfected constructs by Western blot and/or FACS. If 6-well plates were to be used, number of cells and volumes of reagents were halved.

Neisseria gonorrhoeae were suspended in PBS and optical density determined at 550nm.

1x10⁷bacteria were used for infection (MOI 20), which was allowed to proceed for 30-60 minutes as required by experimental set-up and problem posed. Thereafter, medium was exchanged with medium containing 50µg/ml gentamicin for the protection assay (viable, invasive bacteria only), or cells washed three times with medium or PBS for determination of total cell associated bacteria. For Neisseria gonorrhoeae 45 minutes gentamicin were sufficient to kill extracellular bacteria. After washing or incubation with gentamicin, cells were lysed in 1 ml of sterile 1% saponin solution for 15 minutes. A dilution series up to 1x10^-4 was done and 20µl plated on agar in duplicates. The number of colonies, representing viable bacteria, was determined the next day. Dilutions plated were selected by experience.

9.4.7. Transfecting 293T cells via calcium phosphate co-precipitation

The desired amount (1-10µg per 10 cm dish) of plasmid DNA was diluted in 500µl ddH2O. 500µl cold 2xHBS was added and precipitation started by addition of 50µl 2.5M CaCl2 under shaking (vortex). The preparation was incubated for 10-15 minutes at room temperature to allow formation of crystals before pipetting it onto the cells in

Methods

Handling cells

growth medium with 25µM chloroquine. The cells were incubated for 6-8 hours at 37°C before medium was changed to standard growth medium. Expression was allowed to proceed for 48 hours, with the cells being seeded into multi-well plates for experiments 24 hours after transfection.

9.4.8. Preparation for confocal laser scanning microscopy (CLSM)

5x10⁴-1x10⁵ 293T cells were seeded onto coated cover glasses sitting in a 24-well-plate and infected at MOI 20-50 the day after for 30-60 min. Bacteria were either labelled before infection or detected by antibodies after fixation. After infection, cells were washes once with PBS⁺⁺ and fixed with 4% PFA for 20 min at room temperature. PFA was washed away with PBS⁺⁺twice. Staining was conducted as described for bacteria or CLSM respectively.

9.4.9. Whole cell lysates

Whole cell lysates were prepared for control of expression or interaction experiments (immunoprecipitation, pull down). For control of expression, 1x10⁶cells were seeded into 6 cm dishes the day before lysis in 400 µl RIPA-buffer. Cells were scraped, collected in a 1.5 ml tube and genomic DNA sheared with a syringe by drawing the lysate up and down several times. 100 µl sepharose beads were added, and the crude lysate incubated for 10 min under constant inverting at 4°C. Sepharose, along with bound DNA was subsequently collected in a pellet by centrifugation at 14,000xg for 10-20 min. 200 µl of the supernatant were transferred into a clean tube, supplemented with 2xSDS-buffer and frozen down at -20°C.

Lysates to be used in immunoprecipitation or pull-down were extracted from confluent 10 cm dishes using 1 ml triton buffer by default. Depending on the protein complex desired, lysis conditions can be chosen differently. Clearing of the lysate was done as described above and the pure lysate frozen down in aliquots at -80°C.

One aliquot was supplemented with sample buffer and used for expression control via Western blot.