TITLE: Platelet adhesion assay for streptococci
AUTHORS: Samantha King

[DESCRIPTION]
This protocol describes how to perform adhesion assays examining the interaction between streptococci and fixed platelets. 

[GUIDELINES]
Planning the layout of your plate prior to starting the experiment is helpful. The platelets are prepared, and the plate coated as described dx.doi.org/10.17504/protocols.io.n92ld8wb8v5b/v1 in this protocol

[STEPS]
SECTION: Basics
1. The volumes of all washes are 120 µl of PBS 
SECTION: Basics
2. Everything is diluted and made in PBS pH 7.4
SECTION: Basics
3. Binding of each strain or condition is performed in triplicate and each strain has a control of binding to BSA.
SECTION: Preparation of plate
7. Aspirate liquid from platelet coated wells
SECTION: Preparation of plate
8. If you need to pretreat your platelets with neuraminidase follow steps here. If not proceed to step 6.
SECTION: Preparation of plate
8.1. Prepare enough neuraminidase to pre-treat all the necessary wells (50 µl per well).
13. During the 1-hour incubation set up a dilution plate and perform serial dilutions of the inoculums, usually plating 10-1 to 10-4 is adequate.
SECTION: Preparation of plate
8.2. Dilute Neuraminidase (5U/mL) 1:50 in PBS
12. Cover the plate and place it at 37 oC in 5% CO2for 1 hour.
SECTION: Preparation of plate
8.3. Add 50 µl to each well to be treated and 50 µl of PBS to wells that will not be treated
SECTION: Preparation of plate
10. Aspirate BSA, wash the wells twice 120 µl PBS. The plate is ready for adherence. If bacteria are not ready for adherence then leave the wells in PBS until ready to start the assay. However, this time should be minimized. Once inoculums are ready remove the PBS
SECTION: Preparation of plate
9. Block all wells with 120 µl of 3% BSA for 60 min at 37 oC. 
SECTION: Preparation of plate
8.4. Incubate 30 min at 37oC.
SECTION: Preparation of plate
8.5. Wash all wells twice with 120 µl PBS
SECTION: Inoculum preparation
4. Grow bacteria to OD600 0.5 ±0.005 in Todd Hewitt with yeast extract. Place cultures on ice.
SECTION: Inoculum preparation
5. Bacteria are diluted 1:50 in PBS, mix well by pipetting the mixture up and down, this is your inoculum, keep it on ice.
SECTION: Inoculum preparation
6. If you need to add a competitive inhibitor alter the amount of PBS added appropriately. For example, CBM40 is used at a final concentration of 30 µM.
SECTION: Adhesion assay
11. Add 50 µl of inoculum to platelet coated wells and BSA controls

14. After the 1-hour incubation, aspirate the liquid from the wells and wash each well 3x with 120 µl PBS
13.1. Dilute one sample at a time and keep the others on ice
13.2. Mix the samples by pipetting or vortexing immediately prior to dilution.
13.4. Plate 3 x 10 µl spots of each dilution on tryptic soy agar plates with 5% sheep's blood. 
15. Add 100 µl of 0.25% trypsin/1mM EDTA to each well and incubate the plate in 5% CO­2 at 37 oC for 15 min.
16. Using a 200 µl pipette mix the contents over the surface of the well a few times and scrape the pipette tip over the surface to loosen adherent bacteria. Transfer the contents in a micro-centrifuge tube. Place the tube on ice.
17. Perform serial dilutions of samples from test and control wells as described above. Typically, 10-0 to 10-3 is sufficient. Plate the dilutions on TSA Blood agar plates and incubate at in 5% Co 37 oC overnight.
13.3. Perform 10-fold dilutions in PBS in a 96-well plate
13.5. Incubate plate in 5% CO­2 at 37 oC overnight
18. The next day calculate the percentage adherence. Attached is an excel sheet that will help with the calculations - it will need adapting for each experiment.  and determine the percent adherence with
respect to the inoculum. 
18.1. Count the colonies at appropriate dilutions for each inoculum and well. 
18.2. Average the counts for each of the three spots for each inoculum and well. Then calculate the total amount of bacteria in each inoculum and the total number that were bound in each well. 
19. Experiments are conducted on three independent occasions and the percentage adherence for each averaged. The statistical significance is measured by one way ANOVA using Tukey’s post test. p<0.05 is considered significant.
18.3. Average the triplicate wells for each condition and BSA control.
18.4. For each strain subtract the average of the bacteria bound to BSA from the average bound to platelets.