Peripheral blood for flow cytometry was collected by aortic puncture, using a 50 mM EDTA solution as anticoagulant. Erythrocytes were lysed using RBC Lysis Buffer (BioLegend). Total white blood cell count was obtained by preparing a 1:10 dilution of (undiluted) peripheral blood from the orbital sinus using heparin-coated capillary tubes in RBC Lysis Buffer (BioLegend). After organ harvest, single cell suspensions were obtained as follows: for bone marrow, the femur and tibia of one leg were flushed with PBS through a 40-µm nylon mesh. Spleens and lymph nodes were homogenized through a 40-µm nylon mesh, after which erythrocyte lysis was performed using RBC Lysis Buffer (BioLegend). Lungs and liver were cut into small pieces and subjected to enzymatic digestion with 450 U/ml collagenase I, 125 U/ml collagenase XI, 60 U/ml DNase I, and 60 U/ml hyaluronidase (Sigma-Aldrich) for 1 h at 37°C while shaking. Total viable cell numbers were obtained using Trypan Blue (Cellgro; Corning). For selected experiments, the pleural space was lavaged with 2 × 1 ml PBS and the peritoneal space was lavaged with 2 × 5 ml of PBS to retrieve leukocytes. BAL was performed by flushing the lungs with 4 × 1 ml PBS to retrieve the infiltrated and resident leukocytes. Single-cell suspensions were prepared or cells were MACS-sorted according to the manufacturer’s instructions and as described below.
Rbc lysis buffer
Manufactured by BioLegend
Sourced in United States, United Kingdom
RBC lysis buffer is a solution used to selectively lyse or break down red blood cells (RBCs) in a sample. It is commonly used in immunology and flow cytometry applications to remove RBCs and enrich the white blood cell population for further analysis.
Automatically generated - may contain errors
Lab products found in correlation
Dnase 1, by Merck Group (31 mentions)
Gentlemacs dissociator, by Miltenyi Biotec (16 mentions)
Hyaluronidase, by Merck Group (15 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (15 mentions)
Dnase 1, by Roche (14 mentions)
Streptomycin, by Thermo Fisher Scientific (14 mentions)
Cell staining buffer, by BioLegend (12 mentions)
Collagenase d, by Roche (12 mentions)
Percoll, by GE Healthcare (12 mentions)
Penicillin, by Thermo Fisher Scientific (12 mentions)
Lsrfortessa, by BD (11 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (11 mentions)
Ficoll paque plus, by GE Healthcare (11 mentions)
Rpmi 1640, by Thermo Fisher Scientific (11 mentions)
Collagenase 1, by Merck Group (10 mentions)
Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (9 mentions)
Cd45 pe, by BioLegend (9 mentions)
Liberase, by Roche (9 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (9 mentions)
70 μm cell strainer, by BD (9 mentions)
583 protocols using rbc lysis buffer
1
Isolation of Immune Cells from Murine Tissues
2
Multimodal Immune Cell Isolation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Peripheral blood for flow cytometry was collected by aortic puncture, using a 50 mM EDTA solution as anticoagulant. Erythrocytes were lysed using RBC Lysis Buffer (BioLegend). Total white blood cell count was obtained by preparing a 1:10 dilution of (undiluted) peripheral blood from the orbital sinus using heparin-coated capillary tubes in RBC Lysis Buffer (BioLegend). After organ harvest, single cell suspensions were obtained as follows: lungs were cut into small pieces and subjected to enzymatic digestion with 450 U/ml collagenase I, 125 U/ml collagenase XI, 60 U/ml DNase I and 60 U/ml hyaluronidase (Sigma-Aldrich, St. Louis, MO) for 1 h at 37°C while shaking. Lungs were then homogenized through a 40 μm nylon mesh. Lymph nodes were homogenized through a 40-μm nylon mesh, after which erythrocyte lysis was performed using RBC Lysis Buffer (BioLegend). The pleural space was lavaged with 2 × 1 ml of PBS to retrieve leukocytes. Broncho-alveolar lavage (BAL) was performed by flushing the lungs with 4 × 1 ml of PBS to retrieve the infiltrated and resident leukocytes. Single-cell suspensions were prepared. Total viable cell numbers were obtained using Trypan Blue (Cellgro, Mediatech, Inc, VA).
3
Isolation and Enumeration of Rat Splenocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A single-cell suspension of rat spleens was obtained by manual mincing and passing through a 40-μm sterile nylon mesh (Thermo Fisher Scientific, Pittsburgh, PA) with a 3-ml rubber syringe plunger. Cells were collected by centrifugation at 1500 rpm for 3 min. The supernatant was discarded, resuspended in PBS, and centrifuged. The supernatant was discarded, and the cells were resuspended in RBC lysis buffer (BioLegend, San Diego, CA) according to the manufacturer’s method. After removal of RBCs, splenocytes were washed twice with PBS, centrifuged, and resuspended in an appropriate volume of cell staining buffer (BioLegend), and viable cells were counted using the TC20 automated cell counter (Bio-Rad Laboratories, Hercules, CA) using the trypan blue staining method. Approximately 1 ml of rat whole blood was harvested by cardiac puncture using a 1-ml insulin syringe precoated with heparin and collected in tubes containing 100 U/ml heparin. RBC lysis was performed using 1× RBC lysis buffer (BioLegend) according to the manufacturer’s instructions. The cell pellet obtained in the final step was resuspended in staining buffer and viable cells were counted.
4
Isolation and Enumeration of Rat Splenocytes
5
Isolation and Activation of Immune Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CD8+ T cells and CD4+ T cells were isolated from spleens and peripheral lymph nodes, prepared into single cell suspensions and lysed for red blood cells (10x RBC lysis buffer, Biolegend). Negative isolation kits and positive isolation kits (CD11b+ cells) were purchased from StemCell Technologies and isolation was performed following manufacture’s procedures. T cells were activated and cultured as previously described (Jones et al., 2007 (link)) using plate-bound anti-CD3 and anti-CD28 antibodies.
Isolation of intratumoral CD11b+ cells was performed on tumors digested using the tumor preparation protocol (see below). CD11b+ cells were isolated from tumor single cell suspensions using positive isolation kits from StemCell Technologies, following the manufacturer’s protocol.
Macrophages were differentiated from bone marrow flushed using PBS, a 1mL syringe and a 25G needle. Bone marrow was collected, lysed for red blood cells (10x RBC lysis buffer, Biolegend), and plated on non-TC treated plates at 5x106 cells/10cm dish and 20ng/ml of M-CSF (Peprotech).
Cell culture of pancreatic ductal adenocarcinoma cells (PDACs) and ID8 cell lines were maintained in DMEM supplemented with 10% FBS and Pen/Strep.
Isolation of intratumoral CD11b+ cells was performed on tumors digested using the tumor preparation protocol (see below). CD11b+ cells were isolated from tumor single cell suspensions using positive isolation kits from StemCell Technologies, following the manufacturer’s protocol.
Macrophages were differentiated from bone marrow flushed using PBS, a 1mL syringe and a 25G needle. Bone marrow was collected, lysed for red blood cells (10x RBC lysis buffer, Biolegend), and plated on non-TC treated plates at 5x106 cells/10cm dish and 20ng/ml of M-CSF (Peprotech).
Cell culture of pancreatic ductal adenocarcinoma cells (PDACs) and ID8 cell lines were maintained in DMEM supplemented with 10% FBS and Pen/Strep.
6
Immune Cell Isolation from Murine Tissues
7
RBC Lysis and Cell Staining
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
If necessary, the sample was diluted 10× with deionized water, red blood cell (RBC) lysis buffer (BioLegend, San Diego, CA, USA, 420301) was diluted to a 1× working concentration, and then the precipitate was resuspended in 3 mL of 1× RBC lysis buffer. The cells were incubated on ice for 5 min. Cell lysis was stopped by adding 10 mL of cell staining buffer to the tube. The mixture was centrifuged for 5 min at 350× g, and the supernatant was discarded. The wash was repeated as described above.
8
Tumor Immune Cell Profiling Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tumors were minced and digested in HBSS with Collagenase D (1 mg/mL, Roche) or Collagenase A (2mg/ml, Roche) and DNAse I (50 μg/mL, Roche) for 30 minutes at 37°C in a water bath. Digested tissue was then passed through a 70 μm cell strainer, followed by red blood cell lysis with RBC lysis buffer (Biolegend). Single cells were kept on ice.
For whole intratumoral immune cell profiling and DC stainings, CD45-biotin magnetic positive selection (MACS, Miltenyi) was performed to enrich for total tumor immune infiltrate.
For intratumoral T cell stainings, hematopoietic cells were enriched by density gradient centrifugation with 40/70 Percoll (GE Healthcare) for 20 min at 700xg.
Tumor draining lymph nodes (tdLNs) and spleens were minced and digested in RPMI 1640 with Collagenase D (1 mg/mL, Roche) and DNAse I (50 μg/mL, Roche) for 45 minutes at 37°C in a water bath. Digested tissue was then passed through a 70 μm cell strainer and single cells were kept on ice. For spleens, red blood cells were lysed using RBC lysis buffer (Biolegend).
For whole intratumoral immune cell profiling and DC stainings, CD45-biotin magnetic positive selection (MACS, Miltenyi) was performed to enrich for total tumor immune infiltrate.
For intratumoral T cell stainings, hematopoietic cells were enriched by density gradient centrifugation with 40/70 Percoll (GE Healthcare) for 20 min at 700xg.
Tumor draining lymph nodes (tdLNs) and spleens were minced and digested in RPMI 1640 with Collagenase D (1 mg/mL, Roche) and DNAse I (50 μg/mL, Roche) for 45 minutes at 37°C in a water bath. Digested tissue was then passed through a 70 μm cell strainer and single cells were kept on ice. For spleens, red blood cells were lysed using RBC lysis buffer (Biolegend).
9
Isolation of Murine Bone Marrow, Spleen, and Blood Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BM cells were obtained by opening (removing joints) and flushing the hollow bones (femur and tibia), spleens were minced and passed through a cell strainer (70 μm, Falcon, Cat# 352350), and blood was obtained by cardiac puncture. Cells were washed with PBS/2% FCS or R10+ medium (Gibco: 500 mL RPMI1640 Cat# 31870-25; 2mM L-glutamine Cat# 25030-24; 1 mM sodium pyruvate Cat# 11360-039; 100 U/mL penicillin/streptomycin Cat# 15140-122; 0.05μM β-mercaptoethanol Cat# 31350-010; 50 mL heat-inactivated fetal bovine serum (FCS) Cat# 10270-106) and centrifuged at 470g for 7 min at 4°C. Red blood cells (RBC) in the cell pellets from all analyzed organs (except mesenteric lymph nodes) were lysed in RBC lysis buffer (either 0.15 M NH4Cl/0.02 M Hepes solution or RBC lysis buffer from Biolegend, Cat# 420301) for 6 min at RT. The lysis was stopped by adding PBS/2% FCS or R10 medium. Cells were filtered to remove clotted substances (30μm, Sysmex, Cat# 04-0042-2316), centrifuged (470g, 7 min, 4°C), and suspended in the desired volume PBS/2% FCS or R10 medium. Cell concentrations were determined in a Neubauer counting chamber or a Nucleocounter NC3000 (ChemoMetec, Allerod, Denmark).
10
Mouse Hematopoietic Cell Isolation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mouse blood and BM were treated with RBC lysis buffer (BioLegend) before counting and staining for flow cytometry (Table 2 ).
Hematopoietic cell assessment was performed using both hind legs, which were crushed using a pestle and mortar until a homogeneous cell suspension was achieved, or flushed though using a 32G needle. Cells were collected in cold FACS buffer and filtered through a 70-μm nylon strainer (BD Falcon, catalog no. 352340). Cells were treated with RBC lysis buffer (BioLegend) before staining.
Hematopoietic cell assessment was performed using both hind legs, which were crushed using a pestle and mortar until a homogeneous cell suspension was achieved, or flushed though using a 32G needle. Cells were collected in cold FACS buffer and filtered through a 70-μm nylon strainer (BD Falcon, catalog no. 352340). Cells were treated with RBC lysis buffer (BioLegend) before staining.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!