It is mainly used for the testing of research samples.
Operation method
Sample preparation experiments for indirect immunofluorescence labeling by flow cytometry
Principle
Take a certain amount of cell suspension, first add the specific primary antibody, wait for the reaction to be complete and then wash away the unconjugated antibody, in the addition of fluorescently labeled secondary antibody, to generate antigen-antibody complexes, and then detect the fluorescence emitted by the excitation of fluorescein labeled on it by flow cytometry.
Materials and Instruments
Experimental Samples Move I. Treatment of samples from different sources 1 . Cultured cells (1) Cultured cells were digested with 0.25% trypsin. (2) Wash the cells with PBS or saline 2 times, then suspend the cells with PBS or saline, add pre-cooled anhydrous ethanol, the final concentration of 60% - 70%, mix quickly and seal with a sealing film, and place at 4 ℃ can be stored for about 15 days. 2. Fresh specimen (1) Cut the specimen into 1--2mm3 pieces. (2) PBS or saline wash to remove supernatant, add 0.2% collagenase (or 0.15% trypsin) 37 ℃ digested 10 - 30min (according to the experiment and different tissues to determine), and constantly vibration. (3) Filter through 300 mesh nylon sieve to remove tissue clumps, wash with PBS twice, centrifuge at 300g for 5min to obtain digested cells. 3. Paraffin embedded specimen (1) The specimen was cut into 3--5 50μm thick tissue slices on a slicer. (2) The slices were thoroughly dewaxed and hydrated with gradient ethanol (100%, 95%, 70%) and distilled water. (3) 0.5% pepsin (or trypsin) was digested at 37℃ for 30 min, and vibrated every 10 min. (4) 300 mesh nylon sieve filtration, the obtained cell suspension washed twice with PBS and centrifuged at 300xg for 5min. Note: dewaxing must be complete (if you add 100% ethanol without flocculent floating up can be); slice thickness is appropriate, too thin debris, affecting the results of FCM analysis, too thick easy to cause the dewaxing is not clean; pay attention to master the digestion time, to avoid the cells have been released to be digested. Preparation 1. Take 1×106 cells/100μl, add primary antibody first and mix well, and react for 30min at room temperature away from light. 2. 2. Wash the cells with PBS for 2 times, centrifuge to precipitate and discard the supernatant (centrifugation is usually 800--1000rpm for 5min). 3. Resuspend the cells with 100 μl of PBS, add FITC or PE labeled fluorescent secondary antibody (the amount of which should be added according to the instructions) and mix well, and react for 30 min at room temperature. 4. Wash the cells with PBS for two more times, add 500μl PBS to resuspend the cells into single cell suspension, and test on the machine. Caveat 1. The amount of antibody added and the reaction time for the above two staining methods are generally carried out according to the requirements of the instructions for use of the reagents. If it is not stated in the instruction manual, the pre-test should be conducted first to grasp the dose and the optimal reaction time, and then carry out the preparation of flow samples. 2. If the prepared sample cannot be detected on the machine in time, it should be fixed with 1% - 4% paraformaldehyde and can be stored for 5 days at 4℃. Common Problems This method is easy to affect the experimental results because the secondary antibody is usually polyclonal antibody with poor specificity and strong non-specific fluorescent background. Therefore, a negative or positive control should be added when the specimen is prepared. In addition, due to the more steps of the inter-labeling method, it increases the loss of cells and is not suitable for the determination of specimens with a small number of cells. For more product details, please visit Aladdin Scientific website.
Antibody, PBS solution
Pipettes Pipette tips Centrifuge