Harvested bacteria were stained with CFSE and subsequently sub-cultured for 2 hours to reduce the CFSE intensity per bacteria. Human blood (200μl) was infected with 1×107 bacteria and incubated for 30 minutes at 37°C and 5% CO2. Next, blood samples were treated with human Trustain Fcx to block Fc receptors for 10 minutes on ice prior to addition of antibodies. After 30 min incubation, red blood cells were lysed using RBC lysis/Fixation solution (Biolegend) with subsequent incubation for 15 minutes at room temperature. After washing with PBS, cells were analyzed using Amnis Flowsight.
Rbc lysis fixation solution
Manufactured by BioLegend
Sourced in United States
RBC Lysis/Fixation Solution is a laboratory reagent designed to lyse red blood cells and fix remaining cells. It is a ready-to-use solution that can be used as part of sample preparation procedures for flow cytometry or other cell analysis techniques.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2, by BD (3 mentions)
Lymphoprep, by Takeda Pharmaceuticals (1 mentions)
Facscalibur, by BD (1 mentions)
Mouse igg1, by BioLegend (1 mentions)
Human trustain fcx, by BioLegend (1 mentions)
Goat anti rabbit alexa fluor 488 conjugate, by Thermo Fisher Scientific (1 mentions)
Anti cd3 fitc clone ucht1, by BioLegend (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Edta containing tubes, by Greiner (1 mentions)
70 m cell strainer, by Corning (1 mentions)
Cytoflex analyzer, by Beckman Coulter (1 mentions)
Novocyte quanteon flow cytometer, by Agilent Technologies (1 mentions)
Macs buffer, by Miltenyi Biotec (1 mentions)
Lsrfortessa x 20 cell analyzer, by BD (1 mentions)
Cd197 clone g043h7, by BioLegend (1 mentions)
Cd8a clone rpa t8, by BioLegend (1 mentions)
Countbright absolute counting beads, by Thermo Fisher Scientific (1 mentions)
Anti human cd27 clone o323, by BioLegend (1 mentions)
Cd4 clone rpa t4, by BD (1 mentions)
Cd45ra clone hi100, by BD (1 mentions)
15 protocols using rbc lysis fixation solution
1
CFSE Staining and Phagocytosis Assay
2
Phenotyping Human PBMC Subsets
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human peripheral blood mononuclear cells (PBMCs) were collected in heparin-coated tubes and isolated in Lymphoprep™ (Nycomed, Pharma AS, Oslo, Norway) gradients according to the manufacturer's protocol. Red blood cells in the thyroid FNA samples were lysed with RBC lysis/fixation solution (BioLegend, San Diego, CA). Then, the samples were washed with PBS and immunostained with the indicated antibodies. The following antibodies were used: FITC-mouse-anti-human CD4, PerCP-Cy5.5-anti-human CD185 (CXCR5), APC-anti-human CD278 (ICOS), and PE-anti-human CD279 (PD-1) (BioLegend, San Diego, CA). The stained cells were washed with PBS and analyzed by multiparameter flow cytometry (BD FACSCalibur™).
3
Characterization of Meprin β and IL-6R on Human Blood Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Surface expression of meprin β and IL-6R was analyzed on human blood cells. Therefore human heparinized whole blood samples were blocked with Fc block (Human TruStain FcX, BioLegend) diluted 1:100 in FACS buffer (1% BSA [w/vol] in PBS) for 15 minutes and subsequently stained with either anti-meprin β (hEcto1, polyclonal rabbit against human meprin β ectodomain, Pineda) or the pre-immune serum as a negative control, diluted 1:500 in FACS buffer for 1 hour, followed by 30 minutes incubation with the secondary antibody, goat anti-rabbit Alexa Fluor 488 conjugate (Life Technologies), diluted 1:300. For detection of IL-6R and CD3, cells were stained with either anti-IL6R-APC (clone UV4, BioLegend) or the corresponding isotype control (mouse IgG1, κ BioLegend) or anti-CD3-FITC (clone UCHT1, Biolegend) diluted 1:100. All incubations were carried out at 4 °C. Lysis of erythrocytes was performed in RBC Lysis/Fixation Solution (BioLegend) subsequent to staining procedure. Labeled cells were analyzed utilizing a FACS Canto II flow cytometer (BD Biosciences) and data were evaluated with FlowJo (Tree Star) software. T cells were defined as CD3 + whereas granulocytes were gated by FSC/SSC as SSChigh.
All individuals underwent a written, informed-consent process approved by the ethics commission of the Medical Faculty of Kiel University (A 102/14).
All individuals underwent a written, informed-consent process approved by the ethics commission of the Medical Faculty of Kiel University (A 102/14).
4
Flow Cytometry Immunophenotyping of Whole Blood
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole blood was labelled for surface markers with the antibodies and their fluorochromes distributed in four flow cytometry panels named T-cell, B-cell, Tfh–Tγδ cell, and innate immune cell panels (Table S1
5
Multimodal CAR-T Cell Therapy in Nalm-6 Xenograft
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NSG mice were injected via tail vein with 0.5 × 106 cells of a 1:1:1:1 mixture of Nalm-6, hEGFRt-Nalm-6, hHER2t-Nalm-6 and hHER2t-hEGFRt-Nalm-6 cells and 3 days later with 10 × 106 CAR-T cells or mock-T cells, as indicated. Mice were sacrificed 13 days after tumor injection. Bone marrow single-cell suspensions were obtained by flushing one femur with ~10 mL sterile PBS and passing through a 70 µm cell strainer (Corning Falcon). After disruption of the liver through a 70 µm cell strainer and several washing steps, lymphocytes were separated from hepatocytes using a 33.75% Percoll gradient (GE Healthcare). Spleen single-cell suspensions were obtained by two consecutive passages through 70 µm cell strainers. Blood was drawn retro-orbitally from isoflurane-anesthesized mice and collected in EDTA-containing tubes (Greiner). Fifty µL blood was used for the staining with fluorescent antibodies, followed by red blood cell lysis using an RBC lysis/fixation solution (Biolegend).
6
Cell Surface Staining for Flow Cytometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell-surface staining was performed in 100 μL of MACS buffer (Miltenyi Biotec) for 30 minutes on ice in the dark prior to analysis on NovoCyte Quanteon Flow Cytometer (Agilent).
PB was directly stained for 30 minutes on ice in the dark, and then red blood cells were lysed using RBC Lysis/Fixation Solution (BioLegend), according to the manufacturer's instructions. After washing twice, samples were ready for acquisition on a CytoFLEX analyzer (Beckman Coulter).
PB was directly stained for 30 minutes on ice in the dark, and then red blood cells were lysed using RBC Lysis/Fixation Solution (BioLegend), according to the manufacturer's instructions. After washing twice, samples were ready for acquisition on a CytoFLEX analyzer (Beckman Coulter).
7
Multicolor Flow Cytometry Immunophenotyping
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cultured human PBMCs (AllCells, inc.) were analyzed by flow cytometry (MACSquant) to verify the presence of T-cell populations as described (Balazs et al., 2012 (link)) using antibodies against human CD3, CD4, and CD8. Lymphocyte populations were identified by forward and side-scatter profiles and human T-cells were identified by human CD3 staining. Gated human CD3+ T-cell subsets were evaluated for the presence of human CD4 and CD8 T-cells. Mouse whole blood samples were treated with RBC lysis/fixation solution (Biolegend) prior to immunostaining and analysis by flow cytometry as described for cultured huPBMCs.
8
Phenotypic Characterization of Hu-Mouse Immune Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Peripheral blood cells from hu-mice were resuspended in a staining cocktail of anti-human CD27 (clone O323; BioLegend), CD197 (clone G043H7; BioLegend), CD45RA (clone HI100; BD Biosciences), CD8a (clone RPA-T8; BioLegend), CD4 (clone RPA-T4; BD Biosciences), CD3 (clone SK7; BD Biosciences), and CountBright Absolute Counting Beads (for cell quantification; Thermo Fisher). Red blood cells (RBCs) were lysed in RBC lysis/fixation solution (BioLegend), and the remaining cells were fixed with 4% paraformaldehyde. Fixed cells were analyzed by flow cytometry on an LSRFortessa X-20 cell analyzer (BD).
9
Flow Cytometry Analysis of Immune Cells in CKD
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After the proteinuria was reached, whole blood was collected from the tail vein of CKD animals and age matched Sham animals. Red blood cells were lysed with RBC Lysis/Fixation Solution (Biolegend #422401). Fixed cells were labeled with the following antibodies; anti-RP1-BV421 (1:100, BD Biosciences #743053), anti-CD45-BV510 (1:100, Becton Dickinson BV # 740140) anti-CD45RA-APC (1:100, Biolegend #202313), anti-CD3-Alexa488 (1:50, ITK Diagnostics #201406), anti-CD4-APC/Cy7 (1:100, ITK Diagnostics #201518), anti-CD8a-PE-Cy7 (1:100, Antibodychain #25–0084–82), anti-CD11b/c-Percp/Cy5.5 (1:100, ITK Diagnostics #201820), and anti-CD68-PE (1:100, Antibodychain #MA516653). Antibody labeling was performed for 30 min at 4 °C in FACS buffer (10% FCS, 0,1% sodium azide in PBS). Subsequently the cells were washed twice using PBS. Flow cytometric analyses (≥104 events acquired in the “single cell” gate) were performed using a Becton Dickinson FACSCanto II. Gating (supplementary material, Fig. 2) was performed with fluorescence minus one and unstained controls.
10
Flow Cytometry of PEA in Whole Blood
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Flow cytometry of PEA in whole blood was performed as previously described [19 (link)]. Mouse whole blood was harvested by cardiac puncture into tubes containing 3.8 % sodium citrate to prevent coagulation. The whole blood was incubated with phycoerythrin (PE)-conjugated anti-mouse Siglec-F and fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD41 for 30 min at room temperature (RT) in the dark. Next, red blood cells (RBCs) were lysed with the RBC Lysis/Fixation solution (Biolegend, San Diego, CA, USA) for 10 min and washed once with 1× PBS. The cells were analyzed immediately by flow cytometry with the BD FACSCanto II (BD Bioscience, San Diego, CA, USA) as previously described [19 (link)]. A leukocyte gate was set in terms of size and granularity. Within the leukocyte gating, eosinophils were labeled by PE-conjugated anti-mouse Siglec-F. PEA was identified as CD41+ eosinophils (Siglec-F+/CD41+), and at least 1000 events were recorded for each sample. A pooled sample from 3 mice was used to obtain a sufficient number of eosinophils for the analysis.
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!