Antibody binding and deposition of complement components on bacterial surface

FITC labeling of S.pneumoniae Protocol       

Aim: Labeling of bacteria with FITC

Requirements 

Fluorescein isothiocyanate (FITC, isomer I; Sigma Chemicals Co)

DMSO 

PBS1x or Bicarbonate buffer (0.1M NaHCO₃) pH ~8.5;  84mg in 10ml MQ

Assay

1. Prepare 10 mg/ml FITC solution in DMSO (10mg in 1ml DMSO)
2. Grow bacteria in 10 ml THY (~0.6 OD₅₅₀) 
3. Transfer to 15 ml falcon tube and harvest bacteria by centrifugation at 3500 rpm, 10 min.
4. Wash bacteria once with 5 ml bicarbonate buffer (3500 rpm, 10 min) and resuspend in 1 ml bicarbonate buffer. (PBS is also possible)
5. Resuspend bacteria in 1 ml PBS
6. Add 50μl FITC solution (0.5 mg/ml FITC in DMSO) add 50 µl in 1 ml
7. Incubate bacteria for 60 min at 37C and 5% CO₂  (leave the lid a bit open) in the dark.
8. Transfer the 1ml of bacteria to an Eppendorf tube and wash bacteria 2-3 times with PBS or until no free dye can be seen in the supernatant.
9. Resuspend in 1 ml RPMI + 0.05% HSA 
10. Add 10% (v/v) Glycerol-> 110 µl in 1ml 
11. Take 10 µl for cfu counting in blood agar plates
12. Aliquot as quickly as possible and store at -80°C in 100 µl amount (~10⁹ bacteria/ml). Take into account that stocks should keep always protected from light.

Check FITC labeling with FACS

IgG detection

Requirements

-Strain: final concentration 2.5*10^7 bacteria/ml and 15 µl per well.

-Primary antibodies: concentration range from A to H well. Final concentration 8 µg/ml and 15µl per well, start with x2 

-Secondary antibody: 25 µl per well. 

https://www.southernbiotech.com/goat-f-ab-2-anti-human-igg-af647-2042-31

0,5mg/ml stock use 1ug/ml

-Fixation with 1% PFA. 150 μl per well in RPMI-0.05% HAS (stock 10% formaldehyde methanol free Brunschwig 04018-1)

-Round-botton microtiterplate 96 well (Grenier Bio-one) 

- RPMI: Fisher Thermo (Invitrogen) 12017599

Assay IgG detection

- Every well contains 15µ IgG (prepare 16ug/ml FC 8ug/ml and prepare 2 fold dilution range)

- Add 15 µl bacteria to each well 

- Incubate 30 min at 4°C in shaking platform (cold room)

- Add 170 µl buffer (RPMI-0.05% HSA (buffer should be cold, put on ice)

- Centrifugate 7 min at 35000 rpm to wash 

- Suck off the supernatant with the vacuum pomp (do it fast but carefully with the tip bent 45°,   you won’t see the pellet but the bacteria should be on the bottom) 

- Vortex carefully

- Resuspend bacteria with 25 µl of antibody dilution 1 ug/ml

- Incubate 30 min at 4°C in shaking platform (cold room)

- Add 200 µl buffer

- Centrifugate 7 min at 35000 rpm to wash 

- Suck off the supernatant with the vacuum pomp and vortex

- After final wash, add to each well 150 µl 1% PFA

- Keep samples at refrigerator for at least 15 min or overnight

-Determine FL1 mean fluorescence and % positive cells in flow cytometer

Strains used

FITC-labeled bacteria  

Diluted to 5*10^7 b/ml

Antibody concentration range

Final [ ] 8µl/ml and 15 µl/well. Start with 16 µl/ml because it will be diluted x2 with the bacteria vol

Prepare 2-steps dilutions in a different microtiterplate from A to H 

Vol first well(A) 80 µl -> Fill from B to H wells with 40 µl Buffer