Analysis of Immune Cells in Mouse Nervous Tissues Using Flow Cytometry

Abstract

Source: Hidmark, A. S., et al. Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry. J. Vis. Exp. (2017).

This video demonstrates the use of flow cytometry to analyze immune cells in mouse sciatic nerves and dorsal root ganglia (DRGs). The process involves labeling immune cells with fluorophore- and biotin-tagged antibodies, stabilizing and permeabilizing the cells, and using fluorescent markers to detect cell-surface and nuclear characteristics. The technique enables a quantitative assessment of immune cell heterogeneity in nervous tissues.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. Digestion of Sciatic Nerve and DRG

1. Prepare the digestion media (Dulbecco's Modified Eagle Medium [DMEM], 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES], 5 mg/mL bovine serum albumin [BSA], 1.6 mg/mL collagenase Type 4) supplemented with 100 µg/mL of deoxyribonuclease, and store on ice.
   NOTE: The amount of digestion medium required per mouse (sciatic nerves + DRGs) is 1 mL. Once prepared, aliquots of the digestion media can be stored at -20 °C.
2. Transfer the sciatic nerves and DRGs from one mouse into separate 1.5 mL tubes containing 500 µL of digestion media. For the sciatic nerves, chop the material into 15-20 pieces using spring scissors and then incubate the tissue suspensions in a shaking incubator at 37 °C for 20 min with gentle agitation (≤ 450 rpm).
3. Repeatedly triturate the cell suspension through 1,000 µL pipette tip to mechanically dissociate any incompletely digested tissue.
   NOTE: If the suspension is difficult to titrate, then cut the pipette tip with scissors to create a blunt end. Incubate the tissue suspension at 37 °C for 20 min with gentle agitation (≤ 450 rpm).
4. Repeat the trituration through a 1,000 µL pipette tip and incubate for a further 10 min at 37 °C with gentle agitation (≤ 450 rpm).
5. Prepare the FACS buffer (Phosphate-buffered saline [PBS] supplemented with 10% fetal calf serum [FCS] and 1 mM ethylenediamine tetraacetic acid [EDTA]) and store on ice. Rinse and soak a stainless-steel screen with a mesh size of 140 µm with fluorescence-activated cell sorting (FACS) buffer in 60 x 15 mm Petri dish on ice.
   NOTE: Nylon screen filters should be avoided as it leads to a significant loss in the yield of nucleated cells.
6. Repeatedly pass the tissue suspensions through the 140 µm screen filter, as it is held over the Petri dish, with blunt forceps, on ice. Collect the cell suspension from the Petri dish and transfer it to a new 1.5 mL tube stored on ice.
7. Push any incompletely digested tissue that remains on the filter through by repeated trituration with a 1,000 µL pipette tip. Rinse the screen filter twice with 500 µL of FACS buffer and collect the resulting cell suspension in the Petri dish to combine with the cell suspension from step 1.6.
   NOTE: If the flow cytometer used is sensitive to debris, the cell suspension from step 1.7 can be passed through a 70 µm screen filter; however, this may lead to a loss in the yield of nucleated cells.
8. Centrifuge the cell suspensions at 300 x g for 5 min at 4 °C. Discard the supernatant and resuspend the cell pellet in 1 mL of FACS buffer. Repeat this washing step once.
9. Resuspend the cell pellet in 150-350 µL of FACS buffer, depending upon the number of staining that are required (150 µL FACS buffer per staining panel).

2. Staining of Sciatic Nerve and DRG with Fluorescently Labeled Antibodies for Flow Cytometry

1. Transfer 100 µL of the sciatic nerve or DRG cell suspension into a V-shaped well in a 96-well plate on ice.
   NOTE: Single-stained cells are necessary for all antibodies used as they will provide the necessary controls for setting up the flow cytometry with respect to photomultiplier tube (PMT) voltage gains and compensation, as well as to help distinguish specific from non-specific binding.
2. Pool the remaining cells suspensions (approximately 50 µL per sample) from either the sciatic nerves or DRG samples, to serve as an unstained control.
3. Centrifuge the plate at 400 x g for 5 min at 4 °C. Discard the supernatant by inverting the plate over a bin and applying a sharp flick of the wrist, and gently tap dry on paper towels.
4. For surface staining, resuspend each of the cell pellets in 20 µL of FACS buffer containing the antibodies of interest (CD45-A647, CD11b-PerCP/Cy5.5, and major histocompatibility complex [MHC] Class II-Biotin) and incubate, protected from light, on ice for 30 min.
   NOTE: (1) The optimal antibody dilution should be determined prior to use by performing a dilution series according to the manufacturer's product information; (2) Non-specific staining can be reduced by incubation with Fc-block at this step, according to the manufacturer's instructions; and (3) Isotype control staining are generally not required when using primary antibodies directly conjugated with a fluorophore and/or when the antigen(s) of interest produce distinct populations.
5. Add 130 µL of FACS buffer to each well and centrifuge the plate at 400 x g for 5 min at 4 °C. Discard the supernatant as previously described (step 2.3) and wash the cell pellets twice with 150 µL of FACS buffer.
   NOTE: If using an unconjugated primary antibody or a primary antibody conjugated to biotin, then an appropriate secondary reagent, conjugated to a fluorophore, can be added at this point and steps 2.4-2.5 are repeated. In the case of this protocol, streptavidin-PE/Cy7 or Streptavidin-PerCP was used to detect MHC Class II in combination with CD45-A647/CD11b-PerCP/Cy5.5, or in combination with CD68-APC and F4/80-PE/Cy7, respectively.
6. For fixation, after the last washing step, resuspend the cell pellets in 100 µL of 4% paraformaldehyde in PBS (pH 7.4). Incubate the plate on ice for 10 min and then centrifuge at 400 x g for 5 min at 4 °C. Discard the supernatant as previously described (step 2.3) and wash the cell pellets once with 150 µL of FACS buffer.
   Caution: Paraformaldehyde is toxic; wear appropriate protection such as gloves and eyeglasses, etc.
   NOTE: Resuspend the cell pellets in 150 µL of FACS buffer and store protected from the light, at 4° C for up to two days. If no intracellular staining is required, then proceed to step 2.11 after centrifuging the plate at 400 x g for 5 min at 4 °C.
7. For intracellular staining, resuspend the cell pellets in 150 µL of FACS buffer+0.5% detergent, and incubate at room temperature, protected from the light for 15 min. Centrifuge the plate at 400 x g for 5 min at 4 °C. Discard the supernatant as previously described (step 2.3) and wash the cell pellet once with 150 µL of FACS buffer+0.5 % detergent. Resuspend the cell pellet in 20 µL of FACS buffer+0.5% detergent containing the antibodies of interest (CD68-APC, CD206-PE) and incubate, protected from light, at room temperature for 30 min.
8. Add 130 µL of FACS buffer+0.5% detergent to each well and centrifuge the plate at 400 x g for 5 min at 4 °C. Discard the supernatant as previously described (step 2.3) and wash the cell pellets twice with 150 µL of FACS buffer+0.5 % detergent.
   NOTE: If using an unconjugated primary antibody or a primary antibody conjugated to biotin, then an appropriate secondary reagent, conjugated to a fluorophore, can be added at this point and steps 2.9-2.10 repeated.
9. Resuspend the cell pellets in 150 µL of FACS buffer+0.5 % detergent and store, protected from the light, at 4 °C for up to two days.
10. Resuspend the cell pellets with 150 µL FACS buffer+0.5 % detergent supplemented with 5 µg/mL4',6-diamidino-2-phenylindole (DAPI) and incubate for 5 min at room temperature, protected from the light. Centrifuge the plate at 400 x g for 5 min at 4 °C. Discard the supernatant as previously described (step 2.3).
11. Resuspend the cell pellets in 150 µL of PBS and transfer to an appropriately labeled FACs tube. Store at room temperature, protected from the light, until ready for analysis.
    NOTE: After resuspension in PBS, the cells should be analyzed within hours, since the DAPI will slowly dissociate from the DNA, leading to a loss in signal.

3. Setup of Flow Cytometry and Running of Samples

1. Ensure that the flow cytometer is equipped with the appropriate lasers and/or filter sets to measure the fluorophores used to stain the cells. This can be determined under the default cytometer configuration on the system. In the case of this protocol, cells were analyzed using a flow cytometer equipped with three lasers (405 nm, 488 nm, and 633 nm) allowing for the detection of ≤ 5 fluorophores.
2. Select the appropriate channels for detection of fluorophores of interest. All fluorescent signals are best detected with logarithmic amplification.
3. Select an appropriate flow rate for the samples. This parameter can vary depending upon the system used and should be determined empirically. In the case of this protocol, a flow rate of MED-to-HI (35-60 µL/min) was used to prevent clogging of the needle.
4. Select the total number of events/cells to be collected per sample. The minimum number of events should be 1,000,000.
5. Create scatter plots under a global worksheet of the side scatter area (SSC-A )versus forward scatter area (FSC-A), as well for each fluorophore of interest versus FCS-A. Create a statistical view that will display the number of events and the mean fluorescent intensity (MFI) for the selected fluorophores.
6. Set the voltage of the FSC-A and SSC-A channels, such that the smallest cells fit into the lower left 10% of the scatter plot. Determine the background fluorescence and minimum sample fluorescence from the unstained controls from either the sciatic nerves or DRGs. Determine the parent (P1) gate based upon the scatter plot from DAPI-only staining controls.
   NOTE: This is not a collection gate, due in part to the heterogeneous nature of the DAPI+ population in the peripheral nervous system (PNS).
7. Using the FSC scatter plots for each of the fluorophores of interest, adjust the voltages so that the cells of the unstained control fall within the lower quarter of their respective scatter plots. Gates are then created above this region to define those cells that are positive for each of the respective markers/fluorophores. The placement of a gate is confirmed by running of the single-stained control (step 2.1).
   NOTE: Compensation for overlapping emission spectra can be performed at this point or applied retrospectively with the appropriate analysis software.
8. Once the flow cytometer has been set up, as described, the samples can be run.
9. After the run, the remaining sample material can be discarded, and the FCS files can be exported for further analysis.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
C57BL/6 Mouse	Charles River	C57BL/6NCrl
DMEM (+1g/L glucose, Glutamine, Pyruvate)	Thermo	31885023	The source of this material is not important
HEPES	Sigma-Aldrich	H3375
Bovine serum albumin (BSA)	Sigma-Aldrich	A2153
Collagenase Type 4	Worthington Biochemical Corp., US	LS004188
Deoxyribonuclease (DNase) I from bovine pancreas I	Sigma-Aldrich	DN25-1g
Ethylenediaminetetraacetic acid (EDTA)	Sigma-Aldrich	E6758
Foetal Calf Serum (FCS), heat inactivated	Sigma-Aldrich	F4135
Antibody against CD11b-PerCP/Cy5.5	Biolegend	101228	Clone M1/70
Antibody against MHC class II, biotinylated	Biolegend	107603	Clone M5/114.15.2
Antibody against CD45-A647	Biolegend	107603	Clone 30-F11
Streptavidin-PE/Cy7	Biolegend	405206	Used to detect MHCII-biotin with CD45-A647 and CDllb-PerCP/Cy5.5
Streptavidin-PerCP	Biolegend	405213	Used to detect MHCII-biotin with CD68-APC and F4/80-PE/Cy7
Triton-X 100	Sigma-Aldrich	T8787
DAPI	Sigma-Aldrich	D9542-1MG	Dilute in PBS to a 50x and store at 4 °C in the dark
Cell dissociation sieve - tissue grinder kit	Sigma-Aldrich	CD1
V-Shaped, 96-well plates	Greiner/Sigma	M8185
5ml polystryene round-bottom tube, 12x75mm	BD Biosciences	352008
60x15mm petri dish	Greiner/Sigma	Z643084
BD LSR II Flow Cytometer	BD Biosciences