The overall goal of this procedure is to harmlessly and rapidly label the surfaces of living cells or S with bioactive constructs. This is accomplished by first preparing a function spacer, lipid or FSL construct solution. Next, an equal part of the construct solution is mixed with an equal part of the solution of cells or VIRs.
As a third step, the mixture is incubated for 60 minutes at 37 degrees Celsius. The final step is to wash the modified cells or cytes or modified VIRs or VIRs and to use them experimentally as usual. Ultimately, surface modified live cells or S can be visualized through all routine experimental analysis techniques including flow cytometry, microscopy and agglutination.
The main advantage of this technique over existing methods like covalent labeling, is that it is rapid, robust, flexible, and does not affect the vitality of the modified cell or Varian. To prepare the FSL construct stock solution first, add one milliliter of diluent to the product file to reconstitute the dry FSL product, creating a one milligram per milliliter stock solution. Sonicate the vial for 30 seconds, aliquot the stock into 100 microliter sterile containers, and store the containers at two to eight degrees Celsius for up to one week.
To prepare working FSL construct solutions for insertion just prior to use sonicate the solution again for 30 seconds to homogenize any mis cells, then dilute the FSL construct and buffer to the concentration required. Store the working solution at two to eight degrees Celsius for up to a week after Resus suspension in lipid free media or PBS centrifuge the cells for FSL modification free of unbound lipids. Then pack the cells in 100 microliters of diluent, wash the cells for the controls under the same conditions to 100 microliters of unmodified cells.
Add 100 microliters of an appropriate dilution of FSL solution if desired. In parallel, also create negative controls with unrelated FSL solutions and or PBS. Then incubate all the cell subsets for one hour at 37 degrees Celsius after the incubation.
Wash all the cells twice with lipid free media or PBS to remove any free FSL constructs and prepare an appropriate suspension in lipid free media. Once the FSL modification process has been completed, the coys can be stored at four to eight degrees Celsius like a flower. FSL constructs consist of three major components, the functional head or F, which can be a variety of biologically functional groups.
The spacer RS designed to induce spacing of the functional head away from the membrane to improve water dispersity and to be non-reactive with human serum and the DAL lipid or L, which allows the construct to spontaneously incorporate into surfaces four. Representative FSL groups are shown in the following five images. Live murine embryos were directly labeled with FSL fluorescein by the two hours at 37 degrees Celsius method in serum free cell culture media washed and then viewed under fluorescence microscopy.
In this first image, a zop palita free two cell mirroring embryo can be seen. The intense staining in the middle of the embryo is representative of a classic polar body staining. In the next two images, zop palita free four cell and eight cell mirroring embryos that exhibit shadowy staining of the cells that are outside of the microscope P of focus are shown here.
An image of a zop palita free 16 cell mirroring embryo is shown in this last image of FSL fluorescein labeled live mirroring embryos four to five day old intact mirroring blasts embryos with both embryo and zop LUCITA labeled are shown in this next series of images. FSL fluorescein labeling of zebrafish is shown. This first image is of a micro angiography of a 52 hours post fertilization zebrafish larvae that has had FSL fluorescein directly injected into the circulation.
Staining of the zebrafish vasculature can be observed here. FSL fluorescein, heterogeneous zebrafish kidney tissue cells or ZK cytes were created exvivo and then micro injected into the circulation of a 52 hours post fertilization recipient zebrafish in vivo observations of the ZK cytes were made two hours post injection by imaging of the vasculature under fluorescence with time-lapse microscopy shown is a single video frame with large slow moving or immobile cells indicated with orange arrows and fast moving cells, which appeared blurred due to their movement indicated with green arrows. In this image, FSL fluorescein labeling by oral uptake of the construct achieved by immersing zebrafish embryos and FSL fluorescein containing media for up to five days is shown.
The brightfield microscopy of the FSL fluorescein treated zebrafish on the left corresponds to the adjacent fluorescent image on the right. The fluorescence was preferentially located in the intestinal tract. No staining was observed in the untreated control embryos shown here.
Here vesicular stomatitis virus or VSV directly labeled with 10 micrograms per milliliter of FSL fluorescein for two hours at 37 degrees Celsius, followed by fixation with 4%paraform aldehyde, and then analysis by fact scan is shown no purification of the VSV vir. Post FSL labeling was required. This histogram shows swine testicular cells infected with human A Puerto Rico 8 19 34 or H one N one VIRs labeled with FSL fluorescein.
Non fluorescing uninfected cells are represented by the black line while fusion of the H one N one coron with the swine cells, which resulted in fluorescence is indicated by the red line. In this next series of images, the cells were first labeled with FSL biotin for one hour at 37 degrees Celsius, then washed and reacted with Fluor four labeled avadon and then washed and wet mounted for fluorescence microscopy. The first image shows a compiled confocal image of a murine embryo blasts assist, and this figure shows a central confocal slice of the embryo from the preceding image.
Here are live motile humans perm zoa. The blurring occurs as a consequence of their motion. A more distinct representative image of humans spermatozoa fixed in 4%Paraform aldehyde post insertion can be seen in this figure here.
FSL biotin labeled human erythrocytes are shown fixation with 4%paraform aldehyde pres insertion does not affect FSL labeling as illustrated in the following two figures here, 4%parmal fixed RL 95 endometrial human carcinoma cells can be observed. Whereas in this image, the RL 95 endometrial human carcinoma cells are not fixed. Virtually no differences in the labeling are observed between the two images with or without fixation.
FSL biotin labeled RBC cytes observed in a blood sample taken two hours post intravenous infusion of the cytes are shown here on the left, the cells were viewed under light microscopy on the right, the same field of cells was viewed under fluorescence, and the two cytes present can be identified in green. Calculating the ratio of the cytes to unlabeled cells can be used as an indicator of survival. This last FSL biotin labeled cell image shows live human endometrial biotin cytes that were visualized by binding to abated beads.
This final series of images shows FSL construct interactions with blood group markers. The first image is of human red cell GLI cytes coated with a 500 microgram per milliliter concentration of FSLG tested against dilutions of human serum. Human red cells do not naturally react with the Xena antigen GALI antigen.
As such, cytes can be used to quantitate levels of antibody in serum. In this example, the patient was determined to have an antiga titer of one to 32 by creating cytes with decreasing levels of FSL. Similar to creating an antigen titer, an optimal antigen level to detect antibody can be determined, cells can be created to give a positive result only when the antibody level exceeds a specific titer.
The level of FSL antigen required to give a positive result depends on the quality and level of antibody being detected. Typically for carbohydrate antigens, an FSL solution of 100 micrograms per milliliter will result in a strong positive reaction. Human group O red cells modified to have a specific level of a antigen or so-called standardized.
A cytes are used to accurately and reproducibly quantitate antia in human serum. In this example, the cytes were prepared from the donor's own red cells and the antia level in the group O serum tested was found to be one to 32. Shown here in the sixth tube blood group A and B antigens simultaneously inserted into a single group O red cell sample were used to create a week, B week cytes.
These cytes can be used for a VO quality control purposes. The analysis of the specifically formulated a week, B week cyte tested against antia and anti B reagents give the expected weak reactions as evidenced in this figure with the reactions occurring in the middle bottom area of the tubes. This simple technique can be done in two hours if it is performed properly.
