Samples of perfusate (4 ml) were collected into EDTA vacutainers, 0.4 ml dimethyl sulfoxide (DMSO) was added and well-mixed; 2 ml of this solution was then transferred into a cryogenic storage vial, moved to a CoolCell® Cell Freezing Container and stored in a −80°C freezer. Immunophenotyping of the human perfusate samples was performed on a BD LSR II flow cytometer (Becton Dickinson, Oxford, United Kingdom). Leukocytes were identified and gated as CD45+ and their viability assessed using an eFluor™ 506 viability dye (eBioscience, California, USA). Following this, a panel of antibodies was utilized to characterize T helper cells (CD3ε+CD4α+), cytotoxic T cells (CD3ε+CD8α+), double-positive T cells (CD3ε+CD4α+CD8α+), double-negative T cells (CD3ε+CD4α-CD8α-), γδ T cells (γδ+), B cells (CD3ε-CD21+), classical monocytes (CD14+CD163-), non-classical monocytes (CD14+CD163+), immature neutrophils (6D10+2B2-), mature neutrophils (6D10+2B2+), mature eosinophils/basophils (6D10-2B2+), and natural killer cells (CD335+). Cells were treated with red blood cell lysing solution (BD Biosciences, United Kingdom), washed, and resuspended in 0.3 ml of staining buffer. A 20 ml quantity of e123count beads (eBioscience, California, USA) was added and samples were analyzed for 3 min. All gating strategies and analysis were performed using FlowJo version 10.0.6 (12 ).
Red blood cell lysing solution
Manufactured by BD
Sourced in United Kingdom
The Red Blood Cell Lysing Solution is a laboratory reagent designed to lyse or break down the cell membranes of red blood cells. It is a core component in various analytical and diagnostic procedures that require the separation and isolation of cellular components from whole blood samples.
Automatically generated - may contain errors
Lab products found in correlation
Lsr 2 flow cytometer, by BD (2 mentions)
E123count beads, by Thermo Fisher Scientific (2 mentions)
Viability dye efluor 506, by Thermo Fisher Scientific (2 mentions)
Version 10, by FlowJo (1 mentions)
Lsfortessa, by BD (1 mentions)
Phosphate buffered saline (pbs), by Lonza (1 mentions)
35 μm cell strainer, by BD (1 mentions)
Accumax cell dissociation solution, by Innovative Cell Technologies (1 mentions)
70 μm cell strainer, by BD (1 mentions)
Red blood cell lysis buffer, by Merck Group (1 mentions)
Anti cd150 pe cy5 mab, by BioLegend (1 mentions)
Anti c kit apc cy7 mab, by BioLegend (1 mentions)
Hoechst 33342 nucleic acid stain, by Thermo Fisher Scientific (1 mentions)
Lsr 2 sorb, by BD (1 mentions)
Accucheck counting beads, by Thermo Fisher Scientific (1 mentions)
Cytofix cytoperm, by BD (1 mentions)
5 protocols using red blood cell lysing solution
1
Cryopreservation and Immunophenotyping of Human Perfusate
2
Characterization of NK Cell Subsets
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Aliquots of whole blood were stained for 25 min in the dark using the following monoclonal antibodies: CD45 (HI30, APC-eFluor780), CD56 (CMSSB, PE-Cy5), CD3 (UCHT1, Alexa Fluor 700), and CD57 (TB01, FITC), obtained from Thermo Scientific (Wilmington, DE, United States); NKG2C (134591, PE; R&D Systems, MN, United States); and CD16 (3G8, V450; BD Biosciences, San Jose, CA, United States). After staining, the samples were treated with red blood cell lysing solution (BD Biosciences) following the manufacturer’s instructions. Finally, cells were washed twice with phosphate-buffered saline (PBS) (Lonza, Rockland, ME, United States) and suspended in 2% paraformaldehyde. Cells were acquired using LS Fortessa (BD Biosciences, San Jose, CA, United States) and data were analyzed using FlowJo version 10.5.3 (FlowJo, LLC, Ashland, Oregon, United States).
3
Immunophenotyping of Perfusate Samples
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Immunophenotyping of the perfusate samples was performed on a BD LSR II flow cytometer (Becton Dickinson, Oxford, UK). Leukocytes were identified and gated as CD45+ and their viability assessed using an efluor 506 viability dye (ebioscience, San Diego, CA). Following this, a panel of antibodies was utilized to characterize T helper cells (CD3ε+CD4α+), cytotoxic T cells (CD3ε+CD8α+), double-positive T cells (CD3ε+CD4α+CD8α+), double-negative T cells (CD3ε+CD4α-CD8α−), γδ T cells (γδ+), B cells (CD3ε-CD21+), classical monocytes (CD14+CD163−), nonclassical monocytes (CD14+CD163+), immature neutrophils (6D10+2B2−), mature neutrophils (6D10+2B2+), mature eosinophils/basophils (6D10-2B2+), and natural killer cells (CD335+). Cells were treated with red blood cell lysing solution (BD Biosciences, UK), washed, and re-suspended in 0.3 ml of staining buffer. A 20-μl quantity of e123count beads (eBioscience, CA, USA) were added, and samples were analysed for 3 minutes. All gating strategies and analysis were performed using FlowJo version 10.0.6.
4
Dissociation of Extracellular Matrix for Single-Cell Analysis
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The MEM was harvested, as described previously (9 (link)). The harvested MEM was dissociated with collagenase type I (1 mg/mL; catalog number, SCR103; Sigma–Aldrich) and Dispase® II (2 mg/mL; neutral protease, grade II; 4942078001; Sigma–Aldrich) in Dulbecco’s phosphate-buffered saline containing Ca2+ and Mg2+ for 25 min in a 37°C shaking incubator at 100 rpm. The MEM was dissociated further in 1.5 mL of Accumax™ Cell Dissociation Solution (AM105; Innovative Cell Technologies, San Diego, CA, USA) for 8 min in a 37°C shaking incubator at 100 rpm to aid isolation of single cells. Dissociated cells were filtered with 70-μm cell strainers (BD Biosciences, Franklin Lakes, NJ, USA) to eliminate clumps, and incubated in 1 mL of Red Blood Cell Lysing Solution (BD Biosciences) to remove red blood cells. Finally, we utilized 35-μm cell strainers (BD Biosciences) to filter cells and collected them according to manufacturer recommendations. Cell viability >80% was required for subsequent construction of libraries.
5
Bone Marrow and Blood Staining for FACS
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Bone marrow cells were isolated by crushing hind leg bones and (if required) spinal cord in cold RPMI medium, and they were treated with red blood cell lysis buffer (Sigma). Bone marrow was stained with anti-CD135-PE monoclonal antibody (mAb) or anti-CD48-PE mAb (both eBiosciences), or anti-IL-10R-PE mAb (or corresponding isotype control) (BioLegend), anti-CD34-AF700 mAb (eBiosciences), anti-CD150-PE-Cy5 mAb, anti-Sca-1-PE-Cy7 mAb, anti-c-Kit-APC-Cy7 mAb (all BioLegend), and anti-lineage-APC (BD Pharmingen). For proliferation staining, cells were permeabilized with Cytofix/Cytoperm and stained with anti-Ki-67-FITC mAb, corresponding isotype control (all BD Pharmingen), and Hoechst 33342 nucleic acid stain (from Life Technologies).
Blood was stained with anti-B220-PacBlue mAb, anti-CD11b-APC-Cy7 mAb (both BD Pharmingen), anti-Ly6/G-PE-Cy7 mAb (BioLegend), and anti-CD3-AF647 mAb (Caltaq) and treated with red blood cell lysing solution (BD Pharmingen).
Unspecific Fc-receptor binding was blocked by anti-CD16/CD32-unconjugated mAb (BD Pharmingen) prior to surface staining.
Cell counts were determined using AccuCheck counting beads (Life Technologies).
Cells were measured by flow cytometry (FACS) (LSR II Sorb; Becton Dickinson), and data were analyzed with the FlowJo software.
Blood was stained with anti-B220-PacBlue mAb, anti-CD11b-APC-Cy7 mAb (both BD Pharmingen), anti-Ly6/G-PE-Cy7 mAb (BioLegend), and anti-CD3-AF647 mAb (Caltaq) and treated with red blood cell lysing solution (BD Pharmingen).
Unspecific Fc-receptor binding was blocked by anti-CD16/CD32-unconjugated mAb (BD Pharmingen) prior to surface staining.
Cell counts were determined using AccuCheck counting beads (Life Technologies).
Cells were measured by flow cytometry (FACS) (LSR II Sorb; Becton Dickinson), and data were analyzed with the FlowJo software.
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