Jun 27, 2024
Platelet adhesion assay for streptococci
- 1Nationwide Children's Hospital
Protocol Citation: Samantha King 2024. Platelet adhesion assay for streptococci. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg328e1v25/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 27, 2024
Last Modified: June 27, 2024
Protocol Integer ID: 102544
Keywords: Streptococci, platelets, bacterial adhesion
Funders Acknowledgement:
American Heart Association
Grant ID: 19TPA34760750
Disclaimer
We have used this assay for several streptococcal species, but it may need optimizing for different strains.
Abstract
This protocol describes how to perform adhesion assays examining the interaction between streptococci and fixed platelets.
Attachments
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
Safety warnings
Relevant biosafety protocols must be followed.
Basics
Basics
The volumes of all washes are 120 µl of PBS
Everything is diluted and made in PBS pH 7.4
Binding of each strain or condition is performed in triplicate and each strain has a control of binding to BSA.
Inoculum preparation
Inoculum preparation
Grow bacteria to OD600 0.5 ±0.005 in Todd Hewitt with yeast extract. Place cultures on ice.
Bacteria are diluted 1:50 in PBS, mix well by pipetting the mixture up and down, this is your inoculum, keep it on ice.
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.
Preparation of plate
Preparation of plate
Aspirate liquid from platelet coated wells
If you need to pretreat your platelets with neuraminidase follow steps here. If not proceed to step 6.
Prepare enough neuraminidase to pre-treat all the necessary wells (50 µl per well).
Dilute Neuraminidase (5U/mL) 1:50 in PBS
Add 50 µl to each well to be treated and 50 µl of PBS to wells that will not be treated
Incubate 30 min at 37oC.
Wash all wells twice with 120 µl PBS
Block all wells with 120 µl of 3% BSA for 60 min at 37 oC.
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
Adhesion assay
Adhesion assay
Add 50 µl of inoculum to platelet coated wells and BSA controls
Cover the plate and place it at 37 oC in 5% CO2for 1 hour.
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.
Dilute one sample at a time and keep the others on ice
Mix the samples by pipetting or vortexing immediately prior to dilution.
Perform 10-fold dilutions in PBS in a 96-well plate
Plate 3 x 10 µl spots of each dilution on tryptic soy agar plates with 5% sheep's blood.
Incubate plate in 5% CO2 at 37 oC overnight
After the 1-hour incubation, aspirate the liquid from the wells and wash each well 3x with 120 µl PBS
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.
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.
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.
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.
Count the colonies at appropriate dilutions for each inoculum and well.
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.
Average the triplicate wells for each condition and BSA control.
For each strain subtract the average of the bacteria bound to BSA from the average bound to platelets.
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.