To understand which blood cell subtype candidate markers arose from, we investigated their levels in erythrocytes and white blood cells (mononuclear cells and polymorphic nucleated cells). Mononuclear cells were isolated using Ficoll reagent (GE Healthcare, New Jersey, USA). Polymorphic nucleated cells and erythrocyte lysate were isolated from the bottom fraction of Ficoll-spun whole blood. Erythrocyte lysate was prepared using Red Blood Cell Lysis Buffer (Roche, Switzerland). Protocols were carried out based on manufacturer-specific guidelines.
Red blood cell lysis buffer
Manufactured by Roche
Sourced in Switzerland, Germany, United Kingdom, United States
Red blood cell lysis buffer is a solution used to selectively lyse (rupture) red blood cells in a sample, leaving other cell types intact. It is a commonly used tool in cell biology and hematology applications.
Automatically generated - may contain errors
Lab products found in correlation
Dnase 1, by Roche (5 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
Polymorphprep, by Axis-Shield (3 mentions)
Collagenase, by Merck Group (3 mentions)
70 μm cell strainer, by BD (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Pen strep, by Thermo Fisher Scientific (3 mentions)
Gentlemacs dissociator, by Miltenyi Biotec (2 mentions)
Lsrfortessa, by BD (2 mentions)
Vacuette tube, by Greiner (2 mentions)
40 μm cell strainer, by BD (2 mentions)
Hanks balanced salt solution (hbss), by Corning (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Trypan blue, by MP Biomedicals (2 mentions)
Transcriptor first strand cdna synthesis kit, by Roche (2 mentions)
Flowmi tip strainer, by Avantor (2 mentions)
Collagenase p, by Merck Group (2 mentions)
Luna automated cell counter, by Logos Biosystems (2 mentions)
Countess automated cell counter, by Thermo Fisher Scientific (2 mentions)
65 protocols using red blood cell lysis buffer
1
Isolating Blood Cell Subtypes
2
Immunophenotyping of Myeloid Cells in Bone Marrow
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The immunophenotype of the BM myeloid compartment upon exposure to ataluren or analogues was evaluated by flow cytometry as previously reported [2 (link)]. BM samples were depleted of red blood cells by osmotic lysis using the Red Blood Cell Lysis Buffer (Roche, Basel, Switzerland), following the manufacturer’s protocol, and then incubated in IMDM medium (ThermoFisher Scientific, Waltham, MA, USA), supplemented with 20 ng/mL G-CSF (Filgrastim, Hospira, Lake Forest, IL, USA). Cells were incubated with ataluren, or analogues, or vehicle alone (DMSO) for 24 h, then the expression level of CD13, CD33, CD34, CD45, and HLA-DR was evaluated. The possible abnormal expression of mature cell markers (CD11b, CD15) [28 (link)] was checked as well. Flow cytometry analysis was performed using a FACSCanto II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).
3
Characterization of Malignant Effusions
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Primary human malignant ascites and pleural effusion samples were received from the Churchill Hospital, Oxford University Hospitals (Oxford, UK) following informed consent from patients with multiple indications of advanced carcinoma, including but not limited to ovarian, pancreatic, breast and lung. This work was approved by the research ethics committee of the Oxford Centre for Histopathology Research (Reference 09/H0606/5+5). Upon receipt, cellular and fluid fractions were separated and fluid used immediately or aliquots stored at −20°C for future analysis. The cellular fraction was treated with red blood cell lysis buffer (Roche, UK) following manufacturer's instructions. Cell number and viability were determined by trypan blue stain. Cell types present in each sample were determined by antibody staining for EpCAM, EGFR, FAP, CD45, CD11b, CD56, CD3, CD4, CD8, PD1 and CTLA4 and analysed by flow cytometry. For ex vivo T‐cell activation and target cell lysis experiments, fresh cells and fluid were used. In some cases, the adherent cells were passaged in DMEM supplemented with 10% FBS and expanded for later use.
4
Comprehensive Immune Response Analysis
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Mice were euthanized and whole blood, draining lymph nodes, spleens, femurs, tibias, and femoral muscles at the injection site were obtained. Sera were prepared from the blood samples and stored at −80°C until the assays were performed. The spleens were dissociated into cell suspensions using a gentleMACS Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany). The red blood cells were lysed with Red Blood Cell Lysis Buffer (Roche, Basel, Switzerland), and the splenocytes and bone marrow cells were suspended in Roswell Park Memorial Institute-1640 medium (Nacalai Tesque Inc., Kyoto, Japan) supplemented with 10% fetal bovine serum (Sigma-Aldrich, Burlington, MA, USA). The draining lymph nodes were collected, and single-cell suspensions were prepared using BiomusherII (Nippi Research Institute of Biomatrix, Ibaraki, Japan). Femoral muscle samples were fixed with 10% neutral buffered formalin (Mildform 10N; FUJIFILM Wako Pure Chemical, Osaka, Japan) for 24 hours at room temperature (RT), paraffin-embedded, sectioned, and stained with hematoxylin and eosin (H&E).
5
Leukocyte and Erythrocyte Flow Cytometry
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Mouse bone marrow cells were prepared as described above. Splenocytes were isolated as previously described51 (link). For flow cytometry analysis of the leukocytes, red blood cells were depleted with red blood cell lysis buffer (Roche). Cells (0.5–1 × 106) were incubated with Fc block (anti-mouse CD16/32, BioLegend) to block nonspecific Fc binding, stained with isotype control or FITC-anti-mouse CD11b (eBioscience) and PE-anti-mouse Ly-6G/Ly-6C (Gr1) antibody (Biolegend) according to the supplier’s instructions. For flow cytometry analysis of red blood cells, whole mouse bone marrow cells or splenocytes were stained with isotype control or APC anti-mouse Ter119 (BioLegend) and BV-421 anti-mouse CD71 antibody (BD), according to the supplier’s instructions. Flow cytometry was performed using BD LSRFortessa or BD FACSCanto II Flow Cytometer system, followed by analysis with FlowJo software (Tree Star, Inc.).
6
Canine SLAM+ Vero Cells and Ferret WBC
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Vero cells stably expressing the CDV receptor canine SLAM (Vero-cCD150) (kind gift of Dr. Y. Yanagi, Kyushu University, Fukuoka, Japan) were cultured as described previously [64 (link)]. To obtain primary ferret white blood cells (WBC), small-volume blood samples were collected from CDV-naive ferrets in Vacuette tubes (Greiner) containing K3EDTA as an anticoagulant. Red blood cells in blood were subsequently lysed with red blood cell lysis buffer (Roche, Basel, Switzerland), washed and resuspended in complete RPMI 1640 medium (Gibco Invitrogen, Carlsbad, CA, USA) supplemented with 2 mM L-glutamine, 10% (V/V) heat-inactivated fetal bovine serum (FBS), penicillin (100 U/ml) and streptomycin (100 μg/ml). The canine B-cell lymphoma cell line CLBL-1 [41 (link)] (kind gift of Dr. Barbara Rütgen, University of Veterinary Medicine, Vienna, Austria) was grown in complete RPMI-1640 medium supplemented with 10% (V/V) FBS.
7
Dissociation of Cancer Tissue for scGTP-seq
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The cancer tissue was handled within 3 h after excision from the patient. Briefly, the tissue was cut into approximately 1 mm3 pieces in DMEM, and then enzymatically treated with MACS tumour dissociation kit (Miltenyi Biotec, Cat. 130‐095‐929) using 37C_h_TDK_3 program in the gentleMACS Octo Dissociator with Heaters. Dissociated cells, while re‐suspended in DMEM, were subsequently filtered through a 70 μm cell strainer (BD) and centrifuged at 400 g for 10 min at 4°C. After removing the supernatant, the cell pellet was re‐suspended by 1X phosphate‐buffered saline (PBS) with 10% FBS, and the red blood cells were removed using the red blood cell lysis buffer (Roche), according to the manufacturer's instructions. Cells were then filtered through a 40 μm cell strainer (BD), and single cells were picked up by mouth pipetting for scGTP‐seq.
8
Cytokine and T cell analysis
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The serum was separated from peripheral blood by centrifuging at 2000 rpm at 4 °C for 10 min. According to the manufacturer’s instruction, 50 µL of serum samples were applied for analysis of TNF-α, INF-γ, IL-6, IL-12p70, IL-10, and MCP-1 using CBA mouse inflammation kit (BD Biosciences, San Jose, CA, USA, 552364). Data were acquired and analyzed using BD FACVerse flow cytometer and FCAP array software (BD Biosciences, San Jose, CA, USA).
One hundred microliters of peripheral blood were collected and subjected to flow cytometry to determine the differentiation of the CD8+ T cell population. A mixture of fresh blood and 75 µL of PE-conjugated rat-anti mouse CD8a monoclonal antibody (BD Pharmingen, San Jose, CA, USA, 553032) was added to a falcon tube and incubated for 10 min in the dark at RT. The mixture was then incubated for 10 min in 2 mL of red blood cell lysis buffer (Roche, Mannheim, Germany, 11814389001) and 3 mL of PBS was added followed by centrifugation at 380× g for 5 min. The pelleted cells were washed two times with 3 mL of PBS and re-suspended in 0.5 mL PBS. The data were acquired and analyzed using BD FACVerse flow cytometer and BD FACSuite software (BD Biosciences, San Jose, CA, USA).
One hundred microliters of peripheral blood were collected and subjected to flow cytometry to determine the differentiation of the CD8+ T cell population. A mixture of fresh blood and 75 µL of PE-conjugated rat-anti mouse CD8a monoclonal antibody (BD Pharmingen, San Jose, CA, USA, 553032) was added to a falcon tube and incubated for 10 min in the dark at RT. The mixture was then incubated for 10 min in 2 mL of red blood cell lysis buffer (Roche, Mannheim, Germany, 11814389001) and 3 mL of PBS was added followed by centrifugation at 380× g for 5 min. The pelleted cells were washed two times with 3 mL of PBS and re-suspended in 0.5 mL PBS. The data were acquired and analyzed using BD FACVerse flow cytometer and BD FACSuite software (BD Biosciences, San Jose, CA, USA).
9
Isolation of Primary Human Neutrophils
10
Isolation and Purification of Granulosa Cells
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Follicular fluid was aspirated transvaginally under ultrasound guidance during follicle puncture for IVF. After isolation of cumulus-oocyte complexes, follicular fluid was pooled and centrifuged at 1,000 g for 10 minutes. The pellet was retained, and red blood cells were removed by a 60% Percoll gradient and subsequent treatment with red blood cell lysis buffer (Roche, Basel, Switzerland). The remaining cells were purified by centrifugation, suspended in phosphate buffered saline (PBS), and filtered through a mesh (Ted Pella Inc., Redding, CA, USA). The single-cell suspension was layered over 100% fetal bovine serum (Gibco, Grand Island, NY, USA) and centrifuged at 250 g for 15 minutes to remove platelets [27 (link)]. The final pellet was dissolved in Dynal buffer 1 (2 mM ethylenediaminetetraacetic acid, 0.1% bovine serum albumin in PBS) and subjected to immunobead leukocyte depletion.
GCs dispersed in Dynal buffer 1 (2×106 cells/mL) were mixed with 4×107 prewashed paramagnetic beads conjugated with anti-CD45 Ig (Dynal Biotech, Oslo, Norway) at 4℃ for 1 hour. Following capture of the beads with a magnetic separator, the supernatant containing unbound GCs was collected, and the presence of isolated single cells confirmed by microscopic observation (Fig. 1 ). Bead-bound white blood cells were washed with Dynal buffer 1 four times and lysed for RNA isolation.
GCs dispersed in Dynal buffer 1 (2×106 cells/mL) were mixed with 4×107 prewashed paramagnetic beads conjugated with anti-CD45 Ig (Dynal Biotech, Oslo, Norway) at 4℃ for 1 hour. Following capture of the beads with a magnetic separator, the supernatant containing unbound GCs was collected, and the presence of isolated single cells confirmed by microscopic observation (
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