A comprehensive procedure to evaluate the interplay between the complement protein C1q and hyaluronic acid in promoting cell adhesion. We have recently defined that the complement component C1q a molecule of the innate immune system is highly expressed in the tumor and placenta microenvironment and is able to interact with component of the extracellular matrix. In this case, hyaluronic acid.
We propose a method for studying the biological functions of proteins throughout the ecronomic environment. In order to investigate the ability of these molecules to shape the cell division properties of extracellular matrix factors. In order to study how this interaction can be sensed by the cells present in the tissue of the environment, we set up this easy method in which binary cells derived from human tissues were allowed to adhere to C1q bound to extracellular matrix, in this case hyaluronic acid.
For coating procedure the first step is the binding of high molecular weight hyaluronic acid to 96 well tissue coated microplate. For coating, hyaluronic acid is diluted in 0.1 mole carbon buffer PH 9.6 at a concentration of 15 micrograms per milliliter. And gently pipeting is requited to achieve a homogeneous solution.
A 100 microliters of the solution are used to coat the wells. The plate is then transferred at four degrees Celsius overnight. Next day vacuum aspirate the treated wells.
Wash once with dPBS. Add either C1q or BSA in the corresponding wells. C1q is nowadays commercially available at a concentration of one milligram per milliliter and it is diluted at a concentration of 25 micrograms per milliliter.
And dPBS containing 0.5%BSA and 0.7 millimoles calcium and magnesium and divalent cations. BSA LPS free is used at the concentration of 0.5%and date PBF. Plate is then transferred at four degrees Celsius overnight.
C1q dose dependent interactions with immobilized hyaluronic acid is determined by enzyme linked immuno SA.Cells labeling with fast DiI. The lipophilic tracer fast DiI is used to label the cells of interest. Cells resuspended in dPBS are used at the concentration of one million cells per milliliter.
Fast DiI is diluted one to 100 directly into the cell suspension. The cell suspension is mixed manually and transferred at 37 degrees Celsius for 15 minutes. After five minutes incubation, mix the cell suspension and repeat this operation twice more.
To remove the excess of fast DiI, add 10 milliliters of dPBS. Pivot gently up and down and centrifuge at 250 g for seven minutes. Resuspend the cell pellet in one milliliter of human endophilical serum free medium in the presence of 0.1%BSA.
Cells adhesion on hyaluronic acid C1q matrices. Vacuum aspirate the dPBS from the coated wells. Distribute 100 microliters of labeled cell suspension to the coated wells.
200 microliters of labeled cells are taken apart in an ependure tube to be used for standard curve generation. Incubate the plate for 35 minutes at 37 degrees Celsius. Take out the plate from the incubator.
Gently aspirate the unbalanced cells. Wash once with dPBS containing 0.5%BSA and 0.7 millimoles calcium and magnesium. Lys the adherent cells by adding 200 microliters of lysis buffer.
At the same time prepare the standard curve upon one to two serial dilution of the labeled cells in lysis buffer. Perform the reading of the florescence signal at infinite F 200 T-can instrument using 544 nanometers as excitation wavelength and 590 nanometers as emission wavelength. Representative results.
As shown by the representative results, it is evident that cell adhesion on hyaluronic acid found C1q as strongly enhanced in comparison with HA alone or HA bound BSA. The percent of adherent cells on each matrix is calculated on the basis of the standard curve.Conclusions. In our experiments first of all we evaluated the ability of the complement component C12 to bind the extracellular matrix in this case hyaluronic acid using an arisal metal.
Secondly we analyzed the capability of human primary cells stained with fluorescent dye to pursue bound hyaluronic acid. The model that we proposed in this study is an easier faster and cheaper method in alternative to the 3D model of extracellular matrix scaffolds for the evaluation on this cellular response to a combination of stimuli.
