A 100 µL aliquot of whole blood was placed in red blood cell lysis solution (BioLegend, 2.0 mL) for 10 min. The reaction was stopped with the addition of 2.0 mL PBS and centrifuged (2,000 rpm) for 5 min. Afterward, the supernatant was decanted and the leftover pellet was suspended in Cell Staining Buffer (Biolegend) for 15 min. Following an additional centrifugation step, the cell samples were incubated for 15 min on ice with anti-D2R (Millipore) antibodies. The samples were then centrifuged again and incubated for 15 min with anti-CD45 (Invitrogen APC Conjugated, 1:150) and FITC secondary (BD biosciences, 1:150) in the dark and on ice. Sample preparation was finished with an additional centrifugation step and the cells were suspended in 300 µL of Cell Staining Buffer prior to fluorescence-activated cell sorting (FACS) on an Attune Acoustic Focusing Flow Cytometer (Applied Biosystems). Residual samples were plated onto microscope slides and treated with DAPI nuclear stain for visual confirmation of D2R expression on WBCs.
Red blood cell lysis solution
Manufactured by BioLegend
Sourced in United States
Red blood cell lysis solution is a laboratory reagent used to selectively lyse (break down) red blood cells (erythrocytes) in a sample. It is commonly used in flow cytometry and other cell analysis techniques to remove the interference of red blood cells and enrich the population of other cell types for further analysis.
Automatically generated - may contain errors
Lab products found in correlation
Dnase, by Merck Group (4 mentions)
Collagenase 4, by Roche (3 mentions)
Percoll gradient, by GE Healthcare (2 mentions)
Lsrii flow cytometer, by BD (2 mentions)
Dnase 1, by Roche (2 mentions)
Collagenase 4, by Worthington (2 mentions)
Attune acoustic focusing flow cytometer, by Thermo Fisher Scientific (1 mentions)
Cell staining buffer, by BioLegend (1 mentions)
Anti d2r, by Merck Group (1 mentions)
Nk cell isolation kit, by Miltenyi Biotec (1 mentions)
Collagenase 4, by Merck Group (1 mentions)
Fixation permeabilization solution, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa, by BD (1 mentions)
Golgistop, by BD (1 mentions)
Cytofix cytoperm buf kit, by BD (1 mentions)
Ionomycin, by STEMCELL (1 mentions)
Dispase, by Thermo Fisher Scientific (1 mentions)
70 um cell strainer, by BD (1 mentions)
Percoll gradient centrifugation, by GE Healthcare (1 mentions)
Facsaria cell sorter, by BD (1 mentions)
11 protocols using red blood cell lysis solution
1
D2 Receptor Expression in WBCs
2
Spleen Cell Isolation and Preparation
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The spleens were collected and macerated, and the cells were washed twice in DMEM medium containing 10% FBS. Pelleted cells were resuspended in 1 mL of red blood cell lysis solution (BioLegend, San Diego, CA, USA) and then incubated in 1 mL of the FcγR blocking solution.
3
Murine Tumor and Spleen Single-Cell Isolation
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Single-cell suspensions of murine tumors and spleens were prepared as previously described.26 (link) Briefly, murine tumors were minced and digested in complete culture media containing 1 mg/mL of Collagenase IV (Roche) and 50 unit/mL DNase (Sigma) at 37°C with shaking every 10 min. After 30 min of incubation, 10 mL of fresh complete culture media was added and the samples were filtered through a 70 µM filter and centrifuged. Red blood cells were lysed on ice for 5 min with red blood cell lysis solution (BioLegend) followed by addition of complete media. Spleens and lymph nodes were mashed through a 70 µM filter and centrifuged. Red blood cells were lysed as described above, and the cells were washed with PBS, and counted.
4
Isolation and Preparation of Murine Splenic and Tumor Cells
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Spleens were mashed with the blunt end of 3 mL syringe on Petri dishes containing 5 mL of PBS, filtered through 70 μM filter, and centrifuged at 1500 rpm for 5 min at 4 °C. Cell pellets were resuspended in 5 mL of red blood cell lysis solution (BioLegend) on ice for 5 min followed by the addition of 20 mL of complete media. Cells were filtered through 70 μM filter, centrifuged, washed with PBS, and counted. Murine tumors were harvested, minced with scalpel blades, and digested in 2.5 mL of culture media containing 1 mg/mL of Collagenase IV (Roche) and 50 unit/mL DNase (Sigma) in 15 mL tubes at 37 °C with shaking every 10 min. After 45 min of incubation, 10 mL of fresh culture media was added and the samples were filtered through 70 μM filter and centrifuged. Red blood cells were lysed as described above, and the cells were washed with PBS, and counted.
5
Isolation and Co-culture of Liver NPCs and Splenic NK Cells
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Liver non-parenchymal cells (NPCs) were isolated by dissociating MC38 metastatic liver tissue in the presence of collagenase IV (0.1% w/v, Sigma) and DNAse (0.005% w/v, Sigma) for 1 hour before centrifugation on a discontinuous Percoll gradient (GE Healthcare). Isolated NPCs were then used in various assay.
Freshly obtained mouse spleen smashed through 70 µm filter and resuspended in red blood cell lysis solution (BioLegend, San Diego, California, USA). Then splenic NK cells were purified by depletion of magnetically labeled non-NK cells with MACS (Magnetic Activated Cell Sorting) NK Cell Isolation Kit (Miltenyi Biotec, B.G, Germany) according to the manufacturer’s protocol. Purified and enriched NK cells were co-cultured with bone marrow-derived macrophages (BMDMs) in contact or transwell (1:2) in vitro for 12 hours at 37°C and then used for flow cytometry analysis.
Murine M-CSF (Macrophage Colony-Stimulating Factor, 20 ng/mL, PerproTech, New Jersey, USA) in DMEM (Dulbecco's Modified Eagle Medium) supplemented with 10% (v/v) fetal calf serum was used to generate BMDMs in vitro. BMDMs were then replated and cultured in a new 6-well plate overnight for further experiments.16 (link)
Freshly obtained mouse spleen smashed through 70 µm filter and resuspended in red blood cell lysis solution (BioLegend, San Diego, California, USA). Then splenic NK cells were purified by depletion of magnetically labeled non-NK cells with MACS (Magnetic Activated Cell Sorting) NK Cell Isolation Kit (Miltenyi Biotec, B.G, Germany) according to the manufacturer’s protocol. Purified and enriched NK cells were co-cultured with bone marrow-derived macrophages (BMDMs) in contact or transwell (1:2) in vitro for 12 hours at 37°C and then used for flow cytometry analysis.
Murine M-CSF (Macrophage Colony-Stimulating Factor, 20 ng/mL, PerproTech, New Jersey, USA) in DMEM (Dulbecco's Modified Eagle Medium) supplemented with 10% (v/v) fetal calf serum was used to generate BMDMs in vitro. BMDMs were then replated and cultured in a new 6-well plate overnight for further experiments.16 (link)
6
Murine Spleen and Tumor Cell Isolation
7
Multiparameter Flow Cytometry of PBMCs
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Following incubation, whole blood samples were centrifuged (500 g; 3mins) and plasma removed for addition of red blood cell lysis solution (BioLegend) for 20 min at room temperature. Tubes were then centrifuged, and resultant white cells washed twice more in PBS + 1% FBS + 20 mM EDTA before transferring to 96-well plates for staining. For PBMC experiments, tubes were centrifuged after incubation, the media aspirated, and cells transferred to 96-well plates for staining. To stain, plates were centrifuged (500 g; 2mins) and cells resuspended in 50 μl live-dead + surface stain (antibodies detailed in Supplement Table ST1) for 30mins at 4 °C. Cells were then washed twice in 200 μl PBS + 1% FBS + 20 mM EDTA before incubation in 200 μl Fixation/Permeabilization solution (Fisher) for 30mins 4 °C. Cells were washed twice in Permeabilization buffer (PB; Fisher) and resuspended in 50 μl intracellular stain (antibodies detailed in Supplement Table ST1) for 30mins at 4 °C before two final PB washes and final resuspension in 130 μl PBS. Stained cells were stored at 4 °C for 12-48 h prior to flow cytometry.
8
Tumor Single-Cell Suspension Preparation
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The tumors were cut into small pieces, mechanically disrupted and filtered through a 70 µm mesh to generate a single-cell suspension. Dissociated tumor cells were lysed with Red Blood Cell Lysis Solution (No. 420301, Biolegend) and incubated with phorbol 12-myristate 13-acetate (PMA) (No. P1585, Sigma-Aldrich, St. Louis, MO, USA), ionomycin (No. 73724, Stemcell, Vancouver, BC, CAN) and Golgi stop (No. 554715, BD Pharmingen) at 37°C. For cell surface staining, cell suspensions were stained with indicated fluorescent labeled antibodies for 30 min on ice. For intracellular staining, the cells were sorted for fixation and permeabilization using Cytofix/CytoPerm BUF KIT (No. 554714, BD Pharmingen) according to the manufacturer’s guidelines and incubated with primary antibodies. Flow cytometry acquisition was carried out on LSRFortessa (BD Biosciences, San Jose, CA, USA) or a Navios and Gallios (Beckman Coulter, Miami, FL, USA). Compensation beads were used to evaluate spectral overlap. The data were analyzed using FlowJo and CytExpert software according to manufacturers’ instructions.
9
Isolation and Analysis of Myeloid Cells
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Blood and spleen samples from RP mice or controls were lysed by red blood cell (RBC) lysis solution (Biolegend, San Diego, CA). Cells were then passed through 70 um cell strainer (BD Biosciences, Franklin Lakes, NJ) to prepare monolayer cell suspension. Prolapsed or normal rectal tissues were enzymatically digested with enzyme cocktail containing collagenase IV (Worthington Biochemical Corp, Lakewood, NJ), dispase (Gibco), and DNAse I (Roche). Digested cell suspensions were subjected to 40% versus 80% percoll gradient centrifugation (GE Healthcare, Pittsburgh, PA) to enrich leukocytes. To phenotypically analyze immature myeloid cells, cells from blood, spleen, or RP tissue were stained with the following antibodies: PEcy7-CD45 (clone 30-F11), APC-CD11b (clone M1/70), and Percp-Cy5.5-Gr1 (clone RB6-8C5). Multicolor flow cytometry was performed on LSRII flow cytometer (BD Biosciences). All flow antibodies were obtained from Biolegend. Data were analyzed and presented by using Flowjo 9 software (Tree Star, Ashland, OR).
10
Isolation and Characterization of Intestinal Immune Cells
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Intraepithelial lymphocytes (IELs) were prepared from small intestine jejunum by twice 15 min mechanical vibrating in HBSS (Gibco) medium supplemented with 5% FBS, 2 mM EDTA (Invitrogen), 0.15 mg/ml DTT (Sigma-Aldrich), and 10 mM HEPES (Invitrogen). Small intestine Lamina propria (LP), colon and colon tumor tissue were digested respectively with collagenase IV (Worthington Biochemical Corp, Lakewood, NJ), dispase II (Gibco), and DNAse I (Roche). The resulting cells were subjected to 40% and 80% Percoll gradient (GE Healthcare, Pittsburgh, PA) centrifugation to enrich the mononuclear leucocytes. Blood and spleen cells were treated with red blood cell (RBC) lysis solution (Biolegend). Cells from pooled LP and IEL, blood, spleen, and bone marrow cells were passed through a 70 μm cell strainer to prepare monolayer cell suspension for multi-color flow cytometry antibodies staining and analysis. Flow cytometry was performed on LSRII flow cytometer (BD biosciences). Data were analyzed and presented using Flowjo 9 software (Tree Star, Ashland, OR). In some experiments, FCεRIα+c-kit+, CD3+CD4+, CD3+CD8+, or CD11b+Gr1+ cells were sorted from FACSAria cell sorter (BD bioscience) for further experiments.
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