Multi-parametric flow cytometry staining procedure for analyzing tumor-infiltrating immune cells following oncolytic herpes simplex virus immunotherapy in intracranial glioblastoma
2Department of Immunotherapeutics and Biotechnology, School of Pharmacy, Texas Tech University Health Sciences Center, Abilene, TX 79601, USA
3Division of Surgical Oncology, Massachusetts General Hospital, Boston, MA 02114, USA
4Replimune, Inc., Woburn, MA 01801, USA
5Molecular Neurosurgery Laboratory and the Brain Tumor Research Center, Department of Neurosurgery, Massachusetts General Hospital, Boston, MA 02114, USA
6Department of Neurosurgery, Harvard Medical School, Boston, MA 02114, USA
- Dulbecco’s phosphate buffered salt solution (DPBS) (Corning, Cat. # MT21031CV)
- Accutase (Innovative Cell Technologies, Cat. # AT104)
- RQ1 RNase-Free DNase (Promega, Cat. # M6101)
- Heat inactivated fetal bovine serum (ThermoFisher Scientific, Cat. # 10082139)
- MagniSort mouse CD45 positive selection kit (Invitrogen, Cat. # 8802-6865)
- Zombie UV fixable viability kit (BioLegend, Cat. # 423107)
- TruStain FcX (anti-mouse CD16/32) antibody (BioLegend, Cat. # 101319,)
- Alexa Fluor 700 anti-mouse CD45 (BioLegend, Cat. # 103127)
- FITC anti-mouse CD45 (Cat. # 11-0451, Invitrogen)
- APC/Cy7 anti-mouse CD3ε (BioLegend, Cat. # 100329)
- PerCP/Cyanine 5.5 anti-mouse CD4 (BioLegend, Cat. # 100433)
- Brilliant Violet 510TM anti-mouse CD8a (BioLegend, Cat. # 100751)
- Brilliant Violet 605TM anti-mouse/human CD11b (BioLegend, Cat. # 101257)
- Alexa Fluor® 700 anti-mouse Ly-6G/Ly-6C (Gr-1) (BioLegend, Cat. # 108421)
- FITC anti-mouse CD69 (BioLegend, Cat. # 104505)
- PE/Cy7 anti-mouse CD152 (BioLegend, Cat. # 106313)
- Brilliant Violet 421TM anti-mouse CD274 (BioLegend, Cat. # 124315)
- PE/Dazzle 594 anti-mouse CD279 (BioLegend, Cat. # 109115)
- Alexa Fluor® 647 anti-mouse FOXP3 (BioLegend, Cat. # 126407)
- FoxP3 fix/perm buffer set (BioLegend, Cat. # 421403) contains 30 ml 4× concentrated FoxP3 fix/perm buffer (Cat. # 421401) and 25 ml 10× concentrated FoxP3 perm buffer (Cat. # 421402)
- HSV-1 glycoprotein B (gB) HSV-1-specific H-2Kb-HSV-1gB PE-conjugated (Immudex, Cat. # JD2670-PE)
- 15 ml conical centrifuge tubes (Fisher Scientific, Cat. # 12-565-269)
- 50 ml conical centrifuge tubes (Fisher Scientific, Cat. # 12-565-271)
- 1.7 ml microcentrifuge tubes (Fisher Scientific, Cat. # 07-200-534)
- Petri dishes (Fisher Scientific, Cat. # 08-757-100D)
- Disposable scalpel (Fisher Scientific, Cat. # 50-364-913)
- Sorvall ST 16R (Fisher Scientific, Cat. # 75-810-885)
- Sorvall Legend Micro 21R Microcentrifuge (Fisher Scientific, Cat. # 75-002-446)
- Nunc 2 ml serological pipets (Fisher Scientific, Cat. # 12-567-601)
- Nunc 5 ml serological pipets (Fisher Scientific, Cat. # 12-567-602)
- Nunc 10 ml serological pipets (Fisher Scientific, Cat. # 12-567-603)
- Digital water bath (Thomas Scientific, Cat. # 1198Q17)
- 40 µm cell strainer (Fisher Scientific, Cat. # 08-771-19)
- Hemocytometer counting chamber (Fisher Scientific, Cat. # 02-671-10)
- Hemocytometer cover glass (Fisher Scientific, Cat. # 12-519-10)
- Trypan blue stain (Sigma, Cat. # T-8154)
- Binoc inverted light microscope (Fisher Scientific, Cat. # 11-350-119)
- Round bottom 96-well microplates (Fisher Scientific, Cat. # 07-200-99)
- Aluminum foil (Fisher Scientific, Cat. # 01-213-101)
- LSR-II multi-color flow cytometer (BD Biosciences)
|Surface antibodies (with listed vendor concentrations)||Amounta|
|APC/Cy7 anti-mouse CD3ε (0.2 mg/ml)||2.5 µl (or 0.5 µg)|
|PerCP/Cyanine 5.5 anti-mouse CD4 (0.2 mg/ml)||1.25 µl (or 0.25 µg)|
|Brilliant Violet 510TM anti-mouse CD8a (0.2 mg/ml)||2.5 µl (or 0.5 µg)|
|Brilliant Violet 605TM anti-mouse/human CD11b (0.2 mg/ml)||1.25 µl (or 0.25 µg)|
|Alexa Fluor® 700 anti-mouse Ly-6G/Ly-6C (0.5 mg/ml)||0.5 µl (or 0.25 µg)|
|FITC anti-mouse CD69 (0.5 mg/ml)||2 µl (or 1 µg)|
|PE/Cy7 anti-mouse CD152 (0.2 mg/ml)||2.5 µl (or 0.5 µg)|
|Brilliant Violet 421TM anti-mouse CD274||5 µl|
|PE/Dazzle 594 anti-mouse CD279 (0.2 mg/ml)||5 µl (or 1 µg)|
|Surface antibody cocktail/sample||22.5 µl|
1.Harvesting and processing brain tumor samples and magnetic separation of immune cells
1.1.The whole brain bearing tumor is harvested from a sacrificed mouse in 10 ml ice-cold DPBS in a 50 ml falcon tube.
1.2.Decant all the content in a cell culture petri dish and excise the brain tumor quadrant around the needle track wound (site of tumor cell implantation) using a scalpel. Transfer the excised quadrant to a new cell culture petri dish.
1.3.Mechanical dissociation: Cut and mince the brain tumor tissue (tumor size is ~3–4 mm) with scalpel thoroughly for 1–2 min into fragments of about 0.5 mm and smaller.
1.4.Quickly re-suspend the minced tissues in 5 ml ice-cold DPBS and transfer all the contents to a 15 ml falcon tube. Keep the tube on ice.
1.5.Spin samples at 1500 rpm (or 400× g) using Sorvall ST 16R (Fisher Scientific, Waltham, MA) for 5 min at 4°C. Carefully decant off the supernatant to a new falcon tube labeled as “waste”.
1.6.Enzymatic dissociation: Add Accutase (1 ml/tumor quadrant) + DNase I (10 units/ml; to avoid cell clumps) into the tube, re-suspend the pellet using 2 ml pipet, and incubate the solution for 10 min in 37°C digital water bath (Thomas Scientific, Swedesboro, NJ).
1.7.Gently triturate the solution with 2 ml pipet (3–5 times; it will make the remaining tissue pieces even smaller) and add 1% FACS buffer to the tube to a volume of up to 10 ml.
1.8.Transfer and pass this 10 ml cell suspension through a 40 micron cell strainer sitting on top of a new 50 ml falcon tube. Pass another 10 ml ice-cold DPBS through the strainer (in order to wash off any cells that may remain attached to the strainer), which brings a total volume of 20 ml single cell suspension.
1.9.Spin at 1500 rpm (or 400× g) using Sorvall ST 16R for 5 min at 4°C. Carefully decant off the supernatant to the waste tube. Re-suspend the cell pellet in ice-cold DPBS (1 ml DPBS/tumor quadrant) and count the total number of live cells present in single cell suspension.
1.10.Once single cell suspension is prepared, use MagniSort Mouse CD45 Positive Selection Kit (Cat. # 8802-6865, Invitrogen) to isolate CD45+ leukocytes. To do that, follow ‘Experimental Procedure Steps # 1–10’ described in Invitrogen Cat. # 8802-6865. After positive selection, live cells are counted and purity is determined.
1.11.Transfer appropriate number of cells from each sample into new 1.7 ml tubes, including cells for FMO controls (see Notes 3 and 4 in step 1), and proceed to step 2.
2.Blocking Fc receptors, Zombie UV staining (to exclude dead cells) and surface staining
2.1.Spin down the cells (prepared in step 1.11, i.e., 16 sample tubes and 10 FMO controls for 10 antibodies) at 1800 rpm using Sorvall Legend Micro 21R Microcentrifuge (Fisher Scientific, Waltham, MA) for 5 min at 4°C. Carefully decant off the supernatant.
2.2. Re-suspend the cell pellet in 100 µl DPBS/Fc receptor blocking solution and incubate cells on ice for 10 min. Spin down and remove supernatant.
2.3.Add appropriate amount of Zombie UV dye to each tube. Re-suspend and incubate cells for 15 min at room temperature.
2.4.Add appropriate amount of antibody cocktail to each tube to bring to a total volume of 100 µl per sample. Re-suspend the pellet and incubate in the dark for 20 min at room temperature.
2.5.Add 100 µl FACS buffer to each tube and mix gently. Spin down and remove supernatant.
2.6.Repeat step 2.5 one more time and proceed to fixation and intracellular staining.
2.7.TIPS/HINTS: (1) In order to increase efficiency in timing, prepare fresh DPBS/Fc receptor blocking solution while running step 2.1; (2) Similarly, prepare antibody cocktail while running step 2.2, and this step can be extended to 15–20 min, if necessary. Since cells are not fixed, it is important to finish the procedure as quickly as possible; (3) Prepare antibody cocktail just before use, because some flow cytometry antibodies, such as APC/Cy7 and PE/Cy7 do not work well when cocktailed together for a long period of time. Similarly, Brilliant Violets antibodies also do not work well when cocktailed together for a long period of time, such as more than 2–3 h (personal communication with Amy Lee, a flow cytometry expert from BioLegend).
3.Fixation, permeabilization and FoxP3 intracellular staining
3.1.Add 100 µl 1× FoxP3 Fix/Perm solution to each tube, re-suspend the pellet and incubate at room temperature in the dark for 20 min. Spin down (1800 rpm for 5 min, using Sorvall Legend Micro 21R Microcentrifuge) and remove the supernatant.
3.2. Wash once with 100 µl FACS buffer. Spin down and discard the supernatant.
3.3. Add 100 µl FoxP3 Perm buffer, re-suspend the pellet, and incubate cells in the dark for 15 min at room temperature. Spin down and discard the supernatant.
3.4.Again add 100 µl FoxP3 Perm buffer to each tube and re-suspend the pellet.
3.5.Add appropriate amount of intracellular FoxP3 antibody (i.e., 2 µl or 1 µg from 0.5 mg/ml stock for 1 × 106 cells in 100 µl volume, according to manufacturer instructions) to each tube except fluorescent minus FoxP3 tube (FMO-FoxP3) and incubate in the dark for 30 min at room temperature. Spin down and discard the supernatant.
3.6.Wash two times with 100 µl FACS buffer. Spin down and discard the supernatant.
3.7.Re-suspend the pellet in 400 µl FACS buffer and store the samples in the dark in ice until analysis in a flow cytometer (e.g., LSRII; BD Biosciences, San Jose, CA) using appropriate instrument settings.
4.Tetramer staining to determine antigen-specific CD8+ T cells (an optional step during surface staining in step 2)
4.1.Follow steps as described above till step 2.3, and then continue as below.
4.2.After step 2.3, spin samples at 1800 rpm, 5 min at 4°C. Gently decant off the supernatant.
4.3.Re-suspend each sample in 50 µl of tetramer solution and incubate samples in the dark for 45 min at 4°C. Spin down and remove supernatant.
4.4.Re-suspend cells in 100 µl 1% FACS buffer. Spin down and remove supernatant. Repeat this wash step once more time.
4.5.Continue to step 2.4.
|Fluorophores||Laser (excitation wavelength)||Emission maximum (nm)||Filter sets|
|APC/Cy7||Red (633 nm)||785||780/60 (755LP)|
|Alexa Fluor® 700||Red (633 nm)||719||730/45 (685LP)|
|Alexa Fluor® 647||Red (633 nm)||668||670/30|
|PerCP/Cyanine 5.5||Blue (488 nm)||695||695/40 (685LP)|
|FITC||Blue (488 nm)||519||530/30 (505LP)|
|PE/Cy7||Blue (488 nm)||785||780/60 (735LP)|
|PE/Dazzle 594||Blue (488 nm)||610||610/20 (600LP)|
|Brilliant Violet 510||Violet (405 nm)||510||585/42 (550LP)|
|Brilliant Violet 605||Violet (405 nm)||605||675/25 (595LP)|
|Brilliant Violet 421||Violet (405 nm)||421||450/50|
|Zombie UV||Ultraviolet (355 nm)||459||450/50|
|Alexa Fluor® 700||745|
|Alexa Fluor® 647||625|
|Brilliant Violet 510||580|
|Brilliant Violet 605||650|
|Brilliant Violet 421||401|
|1.2, 1.3||Excessive cell death||Tissue left at room temperature Enzymatic dissociation for extended periods||Always keep tissue on ice Enzymatic dissociation should be performed only for 10 min|
|2.4||No extracellular staining||Inappropriate antibody dilution Inadequate incubation time||Use recommended dilution of antibody Use recommended incubation conditions|
|3.2||No intracellular staining||Intracellular staining performed using FACS buffer||Permeabilization buffer should be used for intracellular staining|
|3.7||Spillover of fluorophores||Flow cytometry instrument not setup appropriately||Run recommended Cytometry Setup and Tracking (CST) beads Run compensation controls|
|4.1||No tetramer staining||Mis-match MHC haplotype||Refer to mouse alloantigen database |
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