Peripheral blood was collected from the submandibular vein into heparinized tubes, and the RBCs were lysed using RBC lysis buffer (No. 07850; Stem Cell Technologies). The leukocytes were stained with phycoerythrin (PE)-conjugated anti-murine CD45.1 and allophycocyanin (APC)-conjugated anti-murine CD45.2 (all antibodies from BioLegend) and then evaluated by flow cytometry using a BD Biosciences Canto cytometer. The data were analyzed using the FlowJo software (BD Biosciences), and donor cell engraftment was determined as the percentage of CD45.1+ or CD45.2+ cells present.
Rbc lysis buffer
Manufactured by STEMCELL
Sourced in United States
RBC Lysis Buffer is a solution used to lyse (break down) red blood cells (RBCs) in a biological sample. It is designed to selectively remove RBCs, allowing for the isolation and analysis of other cell types, such as leukocytes (white blood cells). The buffer contains a proprietary formulation that disrupts the RBC membranes without affecting the viability of the remaining cells.
Automatically generated - may contain errors
Lab products found in correlation
Canto cytometer, by BD (1 mentions)
X vivo media, by Lonza (1 mentions)
Ficoll gradient, by STEMCELL (1 mentions)
Cryostor cs10, by STEMCELL (1 mentions)
Collagenase 1, by Merck Group (1 mentions)
Cell strainer, by BD (1 mentions)
Collagenase, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
M3434, by STEMCELL (1 mentions)
7 protocols using rbc lysis buffer
1
Murine Hematopoietic Engraftment Analysis
2
Fetal Bone Marrow Mononuclear Cell Isolation
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Both fetal tibias were completely cleaned of all soft and cartilaginous tissues, placed in a ceramic mortar containing 20 mL ice-cold X-Vivo media (Lonza, Basel, Switzerland), cut into smaller fragments, and manually crushed with a ceramic pestle. The cell suspension and bone fragments were transferred into a 30 μm nylon cell strainer (Thermo Fisher Scientific, Waltham, MA, USA) and rinsed with 10 mL ice-cold X-Vivo media (Lonza). Cell filtrate was subjected to centrifugation at room temperature for 30 min at 400 × g on a Ficoll gradient (Stem Cell Technologies, Vancouver, BC, Canada). FBM mononuclear cells residing at the interface of X-Vivo media and Ficoll were carefully collected, and red blood cells were lysed for 8 min using the RBC lysis buffer ( Stem Cell Technologies). Cells were washed three times with PBS, and CD34+ cells were isolated as described (Robino et al., 2020 (link)) using rhesus macaque-specific antibodies to CD34 (clone 563; BD Pharmingen, San Jose, CA, USA) and the anti-PE MACS magnetic bead system (Miltenyi Biotec, Bergisch Gladbach, Germany ) with the supplied reagents. CD34+ HSPC and CD34− flow-through cell fractions were cryopreserved using CryoStor CS10 (STEMCELL Technologies).
3
Quantifying Metastatic Burden in 4T1 Lung Tumor Model
4
Isolation of Single-Cell from Lung and Lymph Nodes
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To isolate single-cell from the lung tissue, we chopped tissue and incubated at 37℃ with 0.05% trypsin (GIBCO, Grand Island, NY, USA) and 200 unit/mL of collagenase (GIBCO). Ten minutes after incubation, we ground lung tissue with the cell strainer (BD Falcon, Bedford, MA, USA) and incubated at 4℃ with RBC lysis buffer (StemCell Technologies, Vancouver, Canada). For cell isolation from lymphnodes, we ground lymphnode using the cell strainer and incubated with RBC lysis buffer as in the above method.
5
Granulocyte-Colony Stimulating Factor Mobilization
6
Isolation and Xenografting of Circulating Tumor Cells
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Circulating tumour cells (CTCs) were extracted from one EDTA tube of peripheral blood. The blood was first incubated with 50ul RosetteSepTM (cat. no. 15705) per mL of patient blood for 20 min in a rotator at room temperature. After 20 min, blood was diluted with equal volume of wash buffer (WB; 10% HITES media in HBSS) and layered on top of 15 mL of Ficol-Plaque Plus in a 50 mL SepMateTM tube (cat. no. 85450), followed by centrifugation at 1200g for 10 min. Contents above plastic insert of SepMateTM tube were collected in fresh 50mL tube, 30 mL of WB was added, followed by another centrifugation at 300g for 10 min. The supernatant was discarded, and the pellet was resuspended in 3 mL of 1× StemCell Technologies' RBC lysis buffer (cat. no. 20120) and incubated for 10 min at room temperature. Subsequently, 10 mL of WB was added, followed by centrifugation at 300g for 10 min. Finally, the supernatant was removed, and the pellet was resuspended in 100ul of 50% of Matrigel (cat. no. 354230) in HITES media. CTCs in HITES/Matrigel mixture were injected subcutaneously into flank of 6 –16-week immunocompromised (NSG) mice.
7
Cryopreservation of Cord Blood Cells
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Cord blood was collected from the cut end of the umbilical cord attached to the placenta, after the placenta was delivered. A sterile 20 G butterfly needle attached to a 10 ml syringe was inserted into the umbilical cord vein, with the bevel facing up, and 35+/-5 mL blood was collected by negative suction.
The entire fraction of blood was added immediately to a sterile specimen cup treated with sodium heparin for cryopreservation of mononuclear cells and plasma supernatant. The separated plasma was aliquoted into five 0.5 ml trace element-free vials and stored at -80°C.
To isolate mononuclear cells, g cord blood was diluted 1:1 with PBS, then underlaid with 1mL Ficoll for every 4 mL of diluted cord blood, followed by density gradient centrifugation and then treatment in RBC Lysis Buffer (Stem Cell Technologies, #07850) for removal of remaining erythrocytes. Resulting mononuclear cells were cryopreserved in 45% fetal bovine serum + 10% DMSO in RPMI-1640.
The entire fraction of blood was added immediately to a sterile specimen cup treated with sodium heparin for cryopreservation of mononuclear cells and plasma supernatant. The separated plasma was aliquoted into five 0.5 ml trace element-free vials and stored at -80°C.
To isolate mononuclear cells, g cord blood was diluted 1:1 with PBS, then underlaid with 1mL Ficoll for every 4 mL of diluted cord blood, followed by density gradient centrifugation and then treatment in RBC Lysis Buffer (Stem Cell Technologies, #07850) for removal of remaining erythrocytes. Resulting mononuclear cells were cryopreserved in 45% fetal bovine serum + 10% DMSO in RPMI-1640.
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