Flow Cytometry Protocols for Surface and Intracellular Antigen Analyses of Neural Cell Types

The overall goal of this procedure is to analyze the expression of surface and intracellular antigens on neural cells using flow cytometry. This is accomplished by first harvesting neural cells from either in vitro cultures or primary neural tissue. Next, an optional non-specific intracellular stain is applied to the cells.

Then the desired surface antigens are immediately labeled with a primary antibody, whereas the desired intracellular antigens are labeled after cellular fixation and permeation. Ultimately, results can be obtained by using flow cytometric analysis for co-expression of the surface antigens on specific neural subpopulations of interest as defined by intracellular label. This protocol will demonstrate how to prepare tissue culture derived neural cells for interrogation of surface and intracellular antigens with multicolored flow cytometry.

To begin harvest adherent neural cells grown in T 75 flasks by first gently washing the cells once with five milliliters of magnesium and calcium free PBS at room temperature. Then detach the cells from the flask by adding three milliliters of trypsin replacement and incubate at 37 degrees Celsius for two to five minutes. Quench the enzymatic digestion by adding six milliliters of flow buffer after loosening the cell clumps by pipetting the cell suspension up and down a few times, transfer the entire cell suspension into a fresh 15 milliliter tube to isolate the cells.

Place the tube in a centrifuge, spin down the cells for five minutes at room temperature and aspirate the snat in preparation for intracellular dye labeling. Re suspend the cell pellet with five milliliters of PBS plus 0.1%BSA. Otherwise, add five milliliters of flow buffer in conjunction with the standard trian blue staining protocol.

Use a hemo cytometer to estimate the effective viable cell count. Readjust the cell concentration of the existing suspension to 1 million viable cells per milliliter with the same buffer used in the previous step. These cells are now ready for two staining options, a surface antigen antibody stain, or an optional fluorescent intracellular stain with carboxy fluorescein followed by the aforementioned surface antigen stain.

To perform an intracellular labeling step with carboxy fluorescein CIN ester dye first add 10 microliters of CFSE stock solution. For every one milliliter of cell suspension, place the tube on a shaker at 200 RPM and incubate the mixture for five minutes. At room temperature, prevent photobleaching by protecting the tube from direct light exposure.

Quench all unreactive dyes by adding five volumes of serum containing flow buffer and pipette gently up and down a few times to mix. Then spin down the cells for five minutes in a room temperature centrifuge and aspirate the supernatant to remove residual traces of unreactive dyes. Wash the cell pellet by resus suspending it with five volumes of flow buffer.

The cells are then spun down once more in a room temperature centrifuge for five minutes before discarding the SUP natant. Finally, re suspend the cell pellet with flow buffer such that the final cell concentration is 1 million viable cells per milliliter. For a multiplexed experiment, add an equal number of unstained cells of interest from a different experimental condition to the CFSC stained cell suspension.

The cells are now ready for surface antigen tagging with antibodies to begin cell surface staining. Transfer 100 microliters of the cell suspension with or without CFSC labels to a fresh 1.5 milliliter tube. Add fluor four conjugated antibodies to the cell suspension at an amount previously determined by antibody titration tests.

Place the tube on a room temperature orbital shaker at 100 RPMs and incubate the mixture for 30 minutes. In the absence of light, dilute the antibody mixture by adding one milliliter of flow buffer. Gently pipette up and down to mix and spin down the cells in a pre chilled four degrees Celsius micro centrifuge for four minutes.

Aspirate the supinate to remove the unbound antibodies and repeat this wash centrifuge and aspiration. Step one more time. Reus suspend the cell pellet in 100 microliters a flow buffer to the previously stained cells.

Add 500 microliters of the fixation buffer to 100 microliter of the cell suspension. Place the tube on a room temperature orbital shaker at 100 RPMs and incubate for 15 minutes in the dark. The formaldehyde will cross-link all protein and DNA species that are in close proximity with each other and yield non-viable cell suspensions.

Dilute the fixation mixture by adding one milliliter of PBS and gently pipette up and down a few times to mix. Then spin down the fixed cells in a pre chilled four degrees Celsius centrifuge for three minutes. Aspirate the supinate such that 100 microliters of liquid remains above the cell pellet to perme the cell membrane for subsequent intracellular antigen labeling.

Add 500 microliters of permeation buffer to 100 microliters of cell suspension. Place the tube on a room temperature orbital shaker at 100 RPMs and incubate for 15 minutes in the dark. Dilute the permeation mixture by adding one milliliter of PBS gently pipette up and down a few times to mix and spin down the fixed cells in a pre chilled four degree Celsius centrifuge for three minutes.

Aspirate the SUP natant completely prior to incubation. Prepare a primary antibody working stock by diluting the antibody in a PBS plus 0.5%tween 20 buffer. That is further enriched with either 1%BSA 10%donkey serum or 10%goat serum.

Then resuspend the membrane permeable cell pellet with 100 microliters of the primary antibody working stock. Place the tube on a room temperature orbital shaker at 200 RPM and incubate for 30 minutes in the dark. Dilute the primary antibody mixture by adding one milliliter of flow buffer.

Gently pipette up and down a few times to mix and spin down the cells in a pre chilled four degrees Celsius centrifuge for four minutes. Aspirate the supinate completely to remove the unbound antibodies. Next, resuspend the pellet with 100 microliters of the secondary antibody working stock.

Repeat the light protected room temperature incubation for 30 minutes. In the same shaker, dilute the secondary antibody mixture by adding one milliliter of flow buffer. Repeat the same tube inversion mixing, spin down and supernatant removal to eliminate all unbound antibodies.

Repeat the same one milliliter wash centrifugation and supernatant removal steps for two additional times, but with fresh PBS as the wash buffer. Instead, the end result will be a clean cell pellet labeled either with CFSC extracellular or intracellular targeted fluorescent antibodies or any combinations of the three resuspend, the labeled cell pellet in 150 microliter of flow buffer. Transfer the cell suspension to the flow cytometer and commence analysis with the appropriate laser excitation and emission filter settings for each floor of four.

Using the forward and side scatter data as a guide set up initial forward versus side scatter within the flow cytometry analysis software to exclude non-specific data points such as cell debris and dead cells. Next, using raw data from an unstained cell suspension as a negative control, set up multiple fluorescent integrations gates that select for positive emission signals from both extracellular and intracellular antigen labels to account for background fluorescent from multicolor. Spectral overlap effects readjust the fluorescent gates using raw data from single antibody stained controls.

In a multiplexed fax experiment, carboxy fluorescein tags can be used as a baseline signal that could discriminate between cells from two different lineages or experimental conditions without the use of an antibody. For instance, in a heterogeneous cell suspension, one can immediately differentiate between neural cells tagged with CFSC versus untagged fibroblasts just by looking at the presence or absence of fluorescence in the green channel. Furthermore, in conjunction with CFSE Esther labeling, one can use fluorescent antibodies of different colors to determine the differential expression of surface antigens between the CFSE labeled versus unlabeled subsets within a mixed sample.

For instance, SHSY five Y neuroblastoma cells labeled with CFSE in the green fluorescence channel, co-expressed high levels of CD 24 surface antigen, but not of the fibroblast marker CD 54. In contrast, CFSC negative BJ fibroblasts are positive for CD 54 with lower levels of CD 24.Combinatorial. Analysis of neural surface antigens can also be performed in conjunction with antibody-based detection of intracellular antigens.

The inclusion of unstained samples and single stained controls for surface antigens and intracellular antigens is critical as an alternative to primary plus secondary antibody-based labeling of intracellular antigens such as the neural marker map two shown here. Non-covalent fluorescent labeling of the primary antibody can yield comparable results in co-expression analysis and shortens the protocol. After watching this video, you should have a good understanding of how to prepare neural cells for basic flow cytometric analysis using various alternative labeling approaches and combinations thereof.