Data generated using this technique furthers an understanding of autoimmune prone mouse models, as well as humanized mice, that can be used to general antibody or antibody life therapeutics. The availability of reagents with an ever-expanding range of fluorophores, allows for the simultaneous analysis of multiple parameters on individual cells and enables the assessment of B cell heterogeneity. This protocol was developed with the purpose of phenotyping genetically engineered mice.
to determine whether genetic manipulation would alter B cell development. Hi, I'm Faith Harris. I'll be demonstrating the procedure today.
Begin by aliquoting a million of each cell type from each animal into a 96-well U-bottom plate. Make sure sufficient Wells are available for all samples and controls, including full stain, fluorescence-minus-one samples, and unstained samples for each fluorophore. For the bone marrow maturation panel and the spleen maturation panel, aliquot cells into two wells, 1 million cells per well for each full stain sample.
For the single color compensation viability controls, add two million cells each of each cell type to individual wells. Centrifuge the plate at 845 x g for two minutes at four degrees Celsius. Decant the supernatant by quickly inverting and flicking the plate over a sink, taking care not to cross-contaminate the wells.
Wash the cells twice in 200 microliters of DPBS. Centrifuge and decant the supernatant after each wash. Resuspend the cells in 100 microliters of viability dye diluted in DPBS at the ratio of one to 1000, avoiding the cells used for single color compensation.
For each stain set, leave several unstained wells for a completely unstained sample and other controls you might need as well as an additional unstained well for viability FMO control. Add PBS to these wells. Resuspend the cells of single color compensation viability controls in 200 microliters of diluted viability dye.
Transfer 100 microliters of these cells to a 1.5 milliliter microcentrifuge tube and heat it for five minutes at 65 degrees Celsius, then transfer the heated cells back to the original well with the remaining live cells. Incubate the cells at four degrees Celsius for 30 minutes protected from light. After the incubation, centrifuge the cells and decant the supernatant by flicking or inverting the plate without cross-contaminating the other wells.
Wash the cells by resuspending in 200 microliters of DPBS, then centrifuge and decant the supernatant as before. Repeat the DPBS wash again. Add microliters of FC block diluted with stain buffer at a ratio of one to 50 to get a final concentration of 10 micrograms per milliliter.
For peritoneal cells, add five microliters of monocyte blocker to reduce the nonspecific staining. Then, incubate the cells at four degrees Celsius for 15 minutes, protected from light. Prepare full stain master mixes and FMOs in the stain buffer for a final volume of 100 microliters per million cells using the antibody tables in the text manuscript.
Without removing FC block, add 100 microliters of full stain mixes and FMOs to selected wells. Prepare single color compensation controls for each antibody and a stain set. Follow the manufacturer's directions if using compensation beads.
Incubate the cells and the beads at four degrees Celsius for 30 minutes, protected from light. Then, centrifuge and decant the supernatant by inverting and flicking the plate. Wash the cells and beads in 200 microliters of stain buffer three times, centrifuging and decanting the supernatant each time.
Resuspend the cells and beads in 200 microliters of 2%paraformaldehyde in DPBS to fix the samples for analysis. Incubate the cell and beads at four degrees Celsius for 30 minutes, protected from light, then centrifuge and decant the supernatant by inverting or flicking the plate. Resuspend the cells and beads in 200 microliters of stain buffer.
Place a filter plate over a clean 96-well U-bottom plate and transfer each sample to a well of the filter plate using a multi-pipette. Centrifuge the filter plate and decant the supernatant. For the bone marrow and spleen maturation panels, resuspend the fully stained cells in 100 microliters of stain buffer.
Combine the two wells for each animal into one well. Resuspend the remaining panels, FMOs, and controls in 200 microliters of stain buffer. Incubate fixed cells and beads at four degrees Celsius overnight, protected from light.
Record compensation controls for each stain panel using the prepared single stain compensations and set positive and negative gates for each sample. Calculate the compensation matrix using the software. Start acquiring the first sample, making sure that the gates are set appropriately.
Set the machine to run and record the various cell events for each sample as mentioned in the text manuscript. Proceed with data analysis using flow cytometry analysis software, following the gating strategies in the text manuscript. Flow cytometric analysis for characterization of peritoneal B cells in the peritoneum shows the frequencies of viable peritoneal cells, total B cells, B-1 and B-2 subsets, as well as B-1a and B-1b cells in mice.
When bone marrow B cells were analyzed, frequencies of viable cells, total B cells, Fraction A, pre-pro-B cells, Fraction B, Fraction C, Fraction C'Fraction D, immature Fraction E, transitional Fraction E, and fraction F B cells in mice were determined. Analysis of splenic B cells shows the frequencies of viable spleen cells, total B cells, transitional B cells, T1, T2, T3 cells, mature B cells, follicular I cells, follicular II cells, precursor and mature marginal zone cells, and B-1 cells in mice. The frequencies of immunoglobulin kappa and immunoglobulin lambda B cells in mice were determined with flow cytometric analysis of the spleen.
When performing this protocol, you need to resolve signal from one detector bleeding over into the next using compensation controls. These controls could either be cell singly stained or with commercially available compensation beads. Additional assays can be used in conjunction with flow cytometry, like immunohistochemistry to visualize cell localization within lymphoid organs, as well as two photon microscopy to analyze B cell responses in real space and time.
Flow cytometry analysis of the B cell compartments allows for characterization of phenotypes associated with BCR and related deficiencies, perturbations of BCR signaling molecules, or disruption of cytokines that modulate B cell survival.
