Animals were sacrificed and spleens were collected. After dissociating cell clumps, the cell suspension was centrifuged (740g, 10min, RT) and resuspended in 1ml HBSS–EDTA containing 0.5% BSA. Cells were then resuspended in 50% Percoll solution and treated on a three-layer Percoll (Sigma, Cat#GE17–0891-01) gradient (55%, 72%, and 81%) at (1500g, 30min, 10°C without break). LD-PMN-MDSCs were collected from the 50–55% and 55–72% interfaces. Red blood cells (RBCs) were eliminated with RBC lysis solution (Miltenyi, Cat#130–094-183).
Rbc lysis solution
Manufactured by Miltenyi Biotec
Sourced in Germany
RBC lysis solution is a reagent used to selectively lyse or break down red blood cells (RBCs) in a sample. It is a common tool in sample preparation for various cell-based applications, such as flow cytometry and cell separation procedures. The solution's core function is to remove RBCs from a mixed cell population, allowing for the enrichment and analysis of other cell types of interest.
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Lab products found in correlation
Mouse lung dissociation kit, by Miltenyi Biotec (3 mentions)
Percoll, by Merck Group (1 mentions)
1640 medium, by Thermo Fisher Scientific (1 mentions)
Multi tissue dissociation kit, by Miltenyi Biotec (1 mentions)
Cd11b microbead, by Miltenyi Biotec (1 mentions)
Facsmelody cell sorter, by BD (1 mentions)
Stat pack, by Thermo Fisher Scientific (1 mentions)
Zombie nir viability kit, by BioLegend (1 mentions)
Ficoll paque plus, by Greiner (1 mentions)
Pe vio615 anti cd19, by Miltenyi Biotec (1 mentions)
Anti cd3 fitc, by Miltenyi Biotec (1 mentions)
Anti cd4 viogreen, by Miltenyi Biotec (1 mentions)
Anti cd8 apc vio770, by Miltenyi Biotec (1 mentions)
Gentlemacs octo, by Miltenyi Biotec (1 mentions)
Chamber slide, by SPL Life Sciences (1 mentions)
Gentlemacstm dissociator, by Miltenyi Biotec (1 mentions)
Igg2aκ apc, by Thermo Fisher Scientific (1 mentions)
Facsariatm 3 cell sorter, by BD (1 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
10 protocols using rbc lysis solution
1
Isolation of LD-PMN-MDSCs from Spleen
2
Kidney Single-Cell Isolation Protocol
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Each kidney sample from three mice was minced and digested using the Multi Tissue dissociation kit (Miltenyi, 130-110-203), followed by homogenization through syringe-based mechanical disruption. The kidney tissue was enzymatically digested using a mixture of collagenase I, collagenase IV, and hyaluronidase in 1640 medium (Gibco, USA) at 37°C for 40 minutes while suppressing the response with 10% fetal bovine serum (FBS). The resulting dissociated solution was passed through 70-μm cell strainer and then was centrifuged at 400g for 5min at 4°C to collect the cell pellet. To remove any remaining erythrocytes, the red blood cell (RBC) lysis solution (Miltenyi,130-094-183) was applied on ice. Finally, the single-cell suspension was obtained with over 90% viability as detected by Countstar (Alit Biotech, Rigel S2).
3
Isolation of Lymphoid Cell Subsets
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Isolation of CD3ε-, CD11b-, CD11c-, and CD19-positive cells was performed by harvesting the mandibular, accessory mandibular, subiliac, proper axillary, accessory axillary, and medial iliac lymph nodes, as well as the spleen. Tissues were transferred to a 70 µm strainer and homogenized with a plunger. After washing the strainer with MACS buffer (1× PBS, 0.5% bovine serum albumin (BSA), and 2 mM EDTA), the resulting cell suspension was centrifuged at 1000 r.c.f. for 5 min. The supernatant was aspirated and the pellet resuspended in 10 ml RBC lysis solution (Miltenyi Biotec) before incubating for 5 min at room temperature. Cells were centrifuged again at 1000 r.c.f. for 5 min and resuspended in 1 ml MACS buffer, yielding ~1 × 108 cells per ml. Next, the cell suspension was split into two 500 µl fractions, to which 100 µl CD11c or CD11b MicroBeads (Miltenyi Biotec) were added, respectively. The following steps were carried out according to the manufacturer’s instructions for these beads. The flow-through fraction of both purifications was kept and used to isolate CD19- and CD3-positive cells, respectively, by following the manufacturer’s instructions. Purified cells were counted and subsequently pelleted before freezing in liquid nitrogen for storage at −80 °C.
4
Isolation and Migration Assay of Neutrophils and Monocytes
5
Isolation of PBMCs from Human Blood Samples
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Human peripheral blood samples were collected from anonymous healthy donors and patients with MG at Keio University Hospital (Figure 1A
6
Bronchoalveolar Lavage and Blood Collection
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Following PFT measurements, bronchoalveolar lavage (BAL) was performed with three serial instillations of 0.8 mL of sterile PBS through the tracheostomy. Blood samples were obtained through IVC puncture and collected in EDTA tubes. BAL samples were centrifuged at 800×g at 4 °C for 10 min while plasma was separated by centrifugation at 2000×g for 20 min. The BAL and plasma supernatant were flash frozen in liquid nitrogen and saved at − 80 °C for further analysis.
The BAL cell pellet was suspended in 100 μL sterile PBS and 1 mL of RBC lysis solution (Miltenyi Biotec, Auburn, CA). The sample was incubated for 10 min at room temperature and then centrifuged at 300×g for 5 min. The supernatant was discarded and the cells were resuspended in 100 μL sterile PBS and placed on a cytospin slide. Total and differential counts of BAL fluid (BALF) were determined using the May-Grunwald-Giemsa stain (300 cells per animal) as previously described7 (link). Counts were performed in biological duplicates.
The BAL cell pellet was suspended in 100 μL sterile PBS and 1 mL of RBC lysis solution (Miltenyi Biotec, Auburn, CA). The sample was incubated for 10 min at room temperature and then centrifuged at 300×g for 5 min. The supernatant was discarded and the cells were resuspended in 100 μL sterile PBS and placed on a cytospin slide. Total and differential counts of BAL fluid (BALF) were determined using the May-Grunwald-Giemsa stain (300 cells per animal) as previously described7 (link). Counts were performed in biological duplicates.
7
Evaluating Immune Responses in Tumor-Bearing Mice
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CT26 tumor-bearing mice were treated with 50,000 ppm gNO, 20,000 ppm gNO or nitrogen. Fourteen days post gas treatment all tumors were resected. Mouse spleens were dissociated at day 21 post gas treatment with the GentleMACS Octo (Miltenyi-Biotech). Mouse blood samples were processed 21 days post gas treatment using RBC Lysis Solution (cat#130-094-183, Miltenyi-Biotech). Extracellular labeling of T-cells was performed with FITC anti-CD3 (cat#130-119-758, Miltenyi-Biotech), VioGreen anti-CD4 (cat#130-118-693, Miltenyi-Biotech), and APC-Vio770 anti-CD8 (cat#130–120-806, Miltenyi-Biotech) antibodies. Extracellular labeling of B-cells was performed with PE-Vio615 anti-CD19 (cat#130-111-890, Miltenyi-Biotech) antibodies. Extracellular labeling of polymorphonuclear myeloid-derived suppressor cells (MDSCs) was performed with FITC anti-CD3 (cat#130-119-758, Miltenyi-Biotech) and PE-Vio615 anti-CD19 (cat#130-111-890, Miltenyi-Biotech) for negative staining, VioGreen anti-CD11 (cat#130-113-811, Miltenyi-Biotech), PE Ly-6G (cat#130-123-780, Miltenyi-Biotech) antibodies for positive staining and VioBlue anti-Ly6C (cat#130-111-921, Miltenyi-Biotech) for low staining. Samples were analyzed using MacsQuant™ 16 Flow cytometer.
8
Isolation and Analysis of Murine Lung Cell Populations
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Eight-week-old mice were injected with saline or 4 mg/kg BLM, intratracheally. Mice were sacrificed 7 days after BLM injection, and saline perfused lungs were treated using a mouse lung dissociation kit (Miltenyi Biotec, Auburn, CA, USA), in the gentleMACSTM Dissociator (Miltenyi Biotec). Red blood cells (RBCs) were lysed with RBC lysis solution (Miltenyi Biotec), and isolated cells from the mouse lung were double stained with anti-CCSP/anti-EpCAM for club cells or anti-CD74/anti-EpCAM for AT2 cells. Isotype control antibodies (IgG2a κ-FITC and IgG2a κ-APC; eBioscience, San Diego, CA) were used to establish gating parameters for positively stained cells. Double-positive populations were sorted using a BD FACSAriaTM III cell sorter (BD Biosciences, San Jose, CA, USA). Sorted double-positive cells were subjected to an RNeasy Micro Kit (Qiagen, Germany) to obtain mRNA for qRT-PCR assays or plated on chamber slides (SPL Life Sciences, Korea) for in situ proximity ligation assay (PLA) analysis.
9
Single-cell RNA-seq of Mouse Lung
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For single-cell RNA seq (scRNA-seq) of lung tissue derived single cells, whole right lung superior lobe was isolated from each animal. For single cell preparation, a mouse lung dissociation kit (Miltenyi Biotec) was used. Following single cell preparation, cell suspensions were applied to a MACS Smart Strainer (70 μm) and washed twice with 10 ml of DMEM. Cells were pelleted by spinning at 300 x g for a total of 7 minutes at 4°C. Cells were resuspended in 1ml DMEM containing 200 μl DNase (1μg/μl) for 5 minutes at room temperature. Cells were washed by adding 10 ml sterile PBS containing 0.5% BSA and pelleted at 4°C. The RBC lysis was carried out using 1X RBC lysis solution (Miltenyi Biotec; 130-094-183) in a total volume of 1ml for 10 minutes at 4°C. Cells were resuspended in 10 ml chilled DPBS containing 0.5% BSA and pelleted subsequently. Cells were filtered using 35 μm Falcon cell strainer and transferred to a 2 ml lo-binding tube in a total volume of 1ml DPBS containing 0.04% BSA. Cells were subsequently counted using trypan blue dye exclusion assay to determine cell viability and total cell number.
10
Optimized Single-Cell RNA-seq of Murine Lungs
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For single-cell RNA seq (scRNA-seq) of lung tissue derived single cells, whole right lung superior lobe was isolated from each animal. For single cell preparation, mouse lung dissociation kit (Miltenyi Biotec) was used with some additional modifications. Following single cell preparation, cell suspensions were applied to a MACS Smart Strainer (70 μm) and washed twice with 10 ml of DMEM. Cells were pelleted by spinning at 300 × g for a total of 7 minutes at 4°C. Cells were resuspended in 1ml DMEM containing 200 μl DNase (1μg/μl) for 5 minutes at room temperature. Cells were washed by adding 10 ml sterile PBS containing 0.5% BSA and pelleted at 4°C. The RBC lysis was carried out using 1X RBC lysis solution (Miltenyi Biotec; 130-094-183) in a total volume of 1ml for 10 minutes at 4°C. Cells were resuspended in 10 ml chilled DPBS containing 0.5% BSA and pelleted subsequently. Cells were filtered using 35 μm Falcon cell strainer and transferred to a 2 ml lo-binding tube in a total volume of 1ml DPBS containing 0.04% BSA. Cells were subsequently counted using trypan blue dye exclusion assay to determine cell viability and total cell number.
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