This protocol can be used to measure the number of antigenic sites on cellular receptor of interest. The main advantage of this technique is that it produces robust results, even for receptors expressed at a low density. This method enables the evaluation of the reduction of CR1 erythrocyte expression and this is such as Alzheimer's, systemic lupus erythematosus, AIDS, and malaria.
This protocol is useful for any analysis of cell receptor density and can also be applied to the study of cell receptor expression by fluorescence microscope. We recommend spacing out the tubes during the cell and antibody distribution, and to be accompanied by a cytometric specialist during the first analysis if possible. Visual demonstration of the immunostaining and density quantification by flow cytometry is critical for understanding how to properly set the analysis parameters.
Before beginning the analysis, add 250 microliters of sodium EDTA anticoagulated whole blood from blood storage tubes into a 50 milliliter conical tube containing 20 milliliters of four degree Celsius PBS-BSA. Mix the tube contents by gentle inversion and spin the cells by centrifugation. Use a 10 milliliter pipette to discard the supernatant and carefully resuspend the pellet in the residual volume of solution.
Add 20 milliliters of cold PBS-BSA and centrifuge the cells again. At the end of the centrifugation, place the tube in a rack, placed on ice and transfer eight microliters of the washed erythrocytes into a 50 milliliter tube containing three milliliters of PBS-BSA. Then mix the erythrocytes gently by spinning to obtain a homogenous cell suspension.
For erythrocyte immunostaining, carefully transfer 100 microliters of the diluted erythrocytes into individual 1.5 milliliter tubes and collect the red blood cells by centrifugation. After discarding the supernatants, carefully add 20 microliters of a 0.5 microgram per microliter concentration of biotinylated anti-CR1 J3D3 antibody in PBS-BSA, directly to each pellet. Add 20 microliters of PBS-BSA buffer alone to the negative control cells with gentle mixing and incubate the samples for 45 minutes at four degrees Celsius.
At the end of the incubation, wash the samples two times with 750 microliters of fresh PBS-BSA per tube, per wash. After the second wash, add 20 microliters of a one to 10 dilution of streptavidin phycoerythrin in PBS-BSA to each tube with gentle mixing, and incubate the samples for 45 minutes at four degrees Celsius. At the end of the incubation, wash the samples twice, as demonstrated.
After the second wash, fix each cell sample pellet with 450 microliters of fixation buffer during vortexing, before transferring each sample into individual five milliliter round bottom tubes for up to 48 hours of storage at four degrees Celsius. To analyze the immunostained erythrocytes for flow cytometry, click the new experiment button in the flow cytometer, and rename the new experiment. Select forward scatter, side scatter and PE in the cytometer settings window.
In the open experiment, select cytometer settings application settings and create a global worksheet, using the gray boxes and cross hairs to guide the optimization. When all of the parameters have been set, load the unstained control tube onto the cytometer and run the acquisition, optimizing the forward and side scatter voltages to eliminate debris and to ensure that the population of interest is on scale. Next, draw a gate around the red blood cells on the forward versus side scatter plot and display the red blood cell population in the dot plot of PE fluorescence.
If the positive populations are on scale, load the stained control tube onto the cytometer and run the acquisition. To record and analyze samples, unload the stained sample and create a forward versus side scatter plot and a PE fluorescence histogram on a new global worksheet. Load the first sample onto the cytometer, run the acquisition and draw a red blood cell gate around the erythrocytes in the forward versus side scatter plot.
Display the red blood cell population in the PE fluorescence histogram and under the statistics tab, select the mean for the PE fluorescence parameters on red blood cell populations. In the acquisition dashboard, select all of the events in the stopping gate and 10, 000 events to record, and click record data. When the event recording has completed, remove the tube from the cytometer.
The global worksheet plots should look as illustrated. Flow cytometric analysis of immunostained erythrocytes from three subjects of known CR1 densities allows measurement of the mean fluorescence intensity of the labeling for each subject. Plotting a curve using the values of the subject with the known density of erythrocyte CR1 allows these data to be reported as a function of the mean fluorescence intensity.
Comparison of the regression line resulting from this curve to the values of the mean fluorescence intensity of the other subjects allows determination of their CR1 erythrocyte density. Take care that the erythrocytes and antibodies are properly distributed, so the calibration curves of the experimented samples on the negative control are within the range of the cytometer settings. It is possible to adapt this procedure to measure other density cellular receptors, simply by changing the primary antibody and/or adapting the amplification layers.
Whenever you are handling blood, there is a risk of pathogen contamination. Therefore, be sure to use good laboratory practices and to wear the appropriate personal protective equipment.
