The overall goal of this procedure is to prepare a tissue extract that is compatible with functional immunological assays. This is accomplished by first collecting a sample of the tissue of interest. Next, homogenize the tissue sample in a mixture of butanol, aceto, nitrile and water, or BAW.
After determining protein concentration, the extract is allotted and lyophilized to remove solvents. Ultimately, the reconstituted extract is used as a source of antigen in human T-cell proliferation or cytokine secretion assays. The main advantage of this technique over existing methods like detergent lysis, is that deserting extract is free of toxic solvents and detergents.
In this protocol, all human materials should be treated as potentially infectious, and all procedures should be conducted in a class two lamina flow cabinet. Using sterile scissors and forceps, remove fat and fibrous tissue from spleen sections of about one to two centimeters in size. Trim off as much of the outer capsule material as possible.
Cut small pieces of spleen tissue and place each piece into a sterile 50 milliliter Falcon tube Snap. Freeze the pieces of tissue by immersion in liquid nitrogen and store minus 80 degrees Celsius. To begin the procedure for preparing human eyelets for storage culture.
The eyelets in CMRL media at 37 degrees Celsius. Collect eyelets in a 10 milliliter conical bottom tube centrifuge at 1, 500 RPM for five minutes. Discard the media and wash twice in PBS after the second wash.
Pour off the PBS and drain the residual buffer by placing the inverted tube briefly on a paper towel. Be careful not to dislodge the eyelets. Once drained, recap the tube snap freeze in liquid nitrogen and store at minus 80 degrees Celsius.
To prepare a tissue extract, first, remove a tube containing the tissue from minus 80 degrees Celsius and T Thor room temperature. The tissue will be homogenized in a mixture of butanol, acetyl nitrile and water, or BAW that has been previously prepared and stored at four degrees Celsius. Add sufficient ice cold BAW to cover the piece of tissue for the spleen tissue used in this demonstration, used 10 to 20 milliliters of BAW.
Assemble the tissue homogenizer. Clean the homogenizer by homogenizing 20 milliliters of 70%ethanol in water. Next place the homogenizer probe into the tube with the tissue and BAW solution and homogenize the tissue in multiple bursts.
Keep the tube in a nice bucket thereafter to prevent cross-contamination of tissue between samples. Thoroughly clean the homogenizer between samples by homogenizing 10 to 20 milliliters of 70%ethanol in water, followed by BAW buffer centrifuge. The homogenized tissue extract at room temperature.
If an extract containing only soluble material is required, spin at 4, 000 RPM for 10 minutes. If a more crude extract is required, spin up 1000 RPM for five minutes after centrifugation, transfer the supinate to a clean tube and put it on ice. The concentration of protein in the extract is subsequently determined using A BCA or similar assay.
The most important step in this protocol is to accurately measure the protein concentration in the extract so that an appropriate volume of the extract can be lyophilized. To give the desired mass of protein To freeze dry extracts first dilute the homogenate according to the massive protein required and dispense aliquots into labeled five milliliter sterile tubes. In this example, 100 micrograms of protein is allotted per tube.
Using an 18 to 20 gauge sterile needle to make three holes in the cap of each tube freeze tubes by placing them on dry ice for around 10 minutes, switch on the freeze dryer and allow it to equilibrate to minus 100 degrees Celsius. This will take about 30 minutes. When the freeze dryer is ready, place the tubes into the freeze dryer and switch on the vacuum pump.
Depending upon the volume of extract, freeze drying can be completed within three hours. Although samples are routinely left overnight. After completion of the drying cycle, switch off the vacuum pump and slowly allow pressure into the chamber.
Remove the rack of tubes in a sterile hood. Remove the perforated caps from the tubes and replace with new caps. Store tubes at minus 20 degrees Celsius.
The samples can later be reconstituted in culture, media, or other buffers and used in function of biochemical assays. This figure shows the staining of a protein gel loaded with extracts from spleen eyelet depleted pancreatic tissue, and purified human eyelets. These results show a good representation of proteins of different molecular weights for each tissue, the capacity of asana and islet extracts to stimulate human T-cell proliferation was tested using a proliferation assay in which peripheral blood mononuclear cells or PBMC were labeled with the fluorescent dye CFSE cells that proliferated in response to antigen with resultant reduction in CFSE intensity were measured directly by flow cytometry.
The PBMC used in this example were isolated from an individual with type one diabetes. The magnitude of the C cell response was expressed as a ratio of the number of CFSE dim cells per 5, 000 CD four positive CCF SE bright cells without antigen to the number of CFSE dim cells per 5, 000 CD four positive CCF SE bright cells with antigen from triplicate samples. The results show a weak but detectable proliferation in response to Asana with a cell division index or CDI of 3.5.
There was a stronger response to eyelet extract with a CDI of 6.8 inactivated influenza virus was included as a positive control Following this procedure. Other methods like Ali Spot or e Iza can be used to answer additional questions such as what cytokines are secreted in response to tissue derived antigens.
