Sex matched mice at the age of 6–8 weeks were sacrificed by CO2 asphyxiation. Peritoneal cavity cells were collected by rinsing with FACS buffer (1x PBS plus 2% fetal bovine serum). Cells from the bone marrow and spleen were lysed with a red blood cell lysis buffer to remove red blood cells and filtered through a 70 μM cell strainer (BD Biosciences, Franklin Lakes, NJ). After washing with cold 1x PBS, cells were counted by trypan blue staining. One million cells were resuspended in 100 μL FACS buffer and labeled with 1 μL antibodies of B220-APC, CD43-FITC, BP-1-PE, CD24-PerCP/Cy5.5, CD19-APC-Cy7, IgM-PE, IgD-PerCP/Cy5.5, IgM-PErCP/Cy5.5, CD93-APC, CD23-PE, CD21-FITC, CD5-PerCP (BioLegend, San Diego, CA). Cell subpopulations were analyzed on BD LSR II violet at The University of Iowa flow cytometry facility.
Igm pe
Manufactured by BioLegend
Sourced in United States
IgM-PE is a fluorescently labeled antibody that binds to the IgM isotype of immunoglobulins. It is commonly used in flow cytometry applications to detect and quantify IgM-expressing cells.
Automatically generated - may contain errors
Lab products found in correlation
Igd percp cy5, by BioLegend (2 mentions)
70 μm cell strainer, by BD (2 mentions)
Facscanto, by BD (2 mentions)
Igm pe, by BD (2 mentions)
Cd19 apc, by BioLegend (2 mentions)
B220 apc, by BioLegend (1 mentions)
Cd19 apc cy7, by BioLegend (1 mentions)
Cd21 fitc, by BioLegend (1 mentions)
Cd43 fitc, by BioLegend (1 mentions)
Cd24 percp cy5, by BioLegend (1 mentions)
Igm percp cy5, by BioLegend (1 mentions)
Cd93 apc, by BioLegend (1 mentions)
Cd23 pe, by BioLegend (1 mentions)
Kaluza v2, by Beckman Coulter (1 mentions)
Cd11b bv421, by BioLegend (1 mentions)
Fortessa flow cytometer, by BD (1 mentions)
O4 pe, by Miltenyi Biotec (1 mentions)
Glast apc, by Miltenyi Biotec (1 mentions)
Cd45 bv650, by BioLegend (1 mentions)
Facsdiva 8, by BD (1 mentions)
9 protocols using igm pe
1
Murine Immune Cell Characterization
2
Quantification of Brain Cell Populations
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Mouse hippocampi were dissected after CO2 euthanization and gently homogenized through 70 μm cell strainers (#352350, BD Bioscience, United States) in ice-cold PBS with 1% FBS. Homogenates were washed and centrifuged at 500 g for 5 min. Isolated cells were blocked with 10% rat serum in ice-cold PBS for 1 h. Brain cells were stained with 0.5 μL anti-mouse VGLUT2-Alexa488 (#MAB5504A4, Millipore, United States), CD11b-BV421 (#101251, BioLegend, United States), CD45-BV650 (#103151, BioLegend, United States), Glast-APC (#130–123-555, Miltenyi, Germany), and O4-PE (#130–117-357, Miltenyi, Germany) in PBS with 1% FBS on ice for 1 h. Corresponding isotype control antibodies (all BioLegend, United States) included rat IgG2a-Alexa488 (##400,525), IgG2b-BV421 (#400639), IgG2b-BV650 (#400651), IgG2b-APC (#400219), and IgM-PE (#401611). Cells were washed, resuspended in 500 mL PBS, and acquired with a Fortessa flow cytometer (BD Bioscience, United States). Data were analyzed by Kaluza v2.1 software (Beckman Coulter, United States). Astrocytes were defined as Glast+ cells, oligodendrocyte precursor cells (OPCs) as O4+ cells, and microglia as CD45lowCD11bhi cells. Cell populations were calculated as % among total brain cells or microglia as previously described (Piirainen et al., 2021 (link); Chithanathan et al., 2022 (link)).
3
Immunophenotyping of Isolated Cells
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Isolated cells were stained
using specific antibodies (IgM-PE, CD5-APC, BioLegend) in staining
buffer (0.5% bovine serum albumin in phosphate-buffered saline) for
30 min in 4 °C in dark then washed twice. Flow cytometry (FACS)
analysis was performed using FACS Canto (BD Biosciences), and data
were collected using FACSDIva8 (BD Biosciences). FACS data analysis
was done using FlowJo v10.
using specific antibodies (IgM-PE, CD5-APC, BioLegend) in staining
buffer (0.5% bovine serum albumin in phosphate-buffered saline) for
30 min in 4 °C in dark then washed twice. Flow cytometry (FACS)
analysis was performed using FACS Canto (BD Biosciences), and data
were collected using FACSDIva8 (BD Biosciences). FACS data analysis
was done using FlowJo v10.
4
Comprehensive B Cell Immunophenotyping
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Flow cytometric analysis was performed on BD® LSR II Flow Cytometer (Marshallscientific, USA), and data were analyzed with FlowJo10.0 software (Treestar, Ashland, OR, USA). Anti- mouse B220-FITC (103205, Biolegend, USA), CD43-PE-Cy7 (143210, Biolegend, USA), CD24-PE (101807, Biolegend, USA), CD19-PE-Cy7 (552854, BD, USA), CD19-BV421 (115520, Biolegend, USA), CD19-PE (115508, Biolegend, USA), CD69-FITC (104506, Biolegend, USA), CD5-PE (100608, Biolegend, USA), CD86-APC (105011, Biolegend, USA), CD95-PE (152608, Biolegend, USA), BP-1-Alexa Fluor 647 (108312, Biolegend, USA), IgD-APC (405714, Biolegend, USA), IgM-PE (406507, Biolegend, USA), and corresponding isotype control antibodies were purchased from Biolegend. Antibodies were listed in Supplementary Table 3 .
5
Adoptive transfer of irradiated OVA-splenocytes
6
Multiparametric flow cytometry analysis
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Bones and spleens were crushed, cells resuspended and filtered to obtain a single-cell suspension that was analyzed by flow cytometry on a FACSCanto (BD Biosciences). Cells were stained with the following antibodies: CD45-APC, CD10-BV605, CD15-BV605 and glycophorin A-PE (from BD Biosciences) and CD33-BV421, CD19-PerCPCy5.5, CD34-APCCy7, CD68-PE, CD14-BV605, CD117-PECy7, IgM-PE, CD3-PECy7, FceRI-PE and CD25-BV421 (from BioLegend, San Diego, CA, USA). Control cells were stained with matching isotype controls. Sorting of cells was performed on a FACSAria (BD Biosciences).
For intracellular staining of phosphorylated STAT5 (signal transducer and activator of transcription 5), sorted pre-B cells were fixed in 1.6% paraformaldehyde for 10 min at room temperature. Cells were stored in 90% ethanol in −80 °C until analysis. Cells were washed two times in ice cold phosphate-buffered saline before resuspension in phosphate-buffered saline with 2% fetal calf serum. Cells were kept on ice and stained with antibodies against phosphorylated STAT5 (STAT5P-Alexa Flour 647 from BD) or matching isotype controls. Levels of phosphorylated STAT5 are presented as median fluorescence intensity, normalized to the median fluorescence intensity of isotype-stained cells.
For intracellular staining of phosphorylated STAT5 (signal transducer and activator of transcription 5), sorted pre-B cells were fixed in 1.6% paraformaldehyde for 10 min at room temperature. Cells were stored in 90% ethanol in −80 °C until analysis. Cells were washed two times in ice cold phosphate-buffered saline before resuspension in phosphate-buffered saline with 2% fetal calf serum. Cells were kept on ice and stained with antibodies against phosphorylated STAT5 (STAT5P-Alexa Flour 647 from BD) or matching isotype controls. Levels of phosphorylated STAT5 are presented as median fluorescence intensity, normalized to the median fluorescence intensity of isotype-stained cells.
7
Phenotypic Analysis of Freshly Isolated PBMCs
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The phenotype of freshly isolated PBMCs were analyzed by flow cytometry. The following fluorochrome-conjugated mAbs and corresponding isotype control antibodies were used: APC-cy7-CD3 (clone: UCHT1; Biolegend), Percp-cy5.5-CD14 (clone: M5E2; Biolegend), Percp-cy5.5-CD19 (clone: HIB19; Biolegend), APC-CD16 (clone: 3G8; Biolegend), PE-cy7-CD56 (clone: HCD56; Biolegend), PE-CD160 (clone: BY55; Biolegend), PE-IgM (clone: MM-30; Biolegend), Brilliant Violet 421-CD69 (clone: FN50; Biolegend), Brilliant Violet 421-IgG1 (clone: MOPC-21; Biolegend), Alexa Fluor® 488-GLUT1 (clone: 202915; R&D Systems), PE-CD36 (clone: CB38; BD PharMingen™), PE-IgM (clone: G155-228; BD PharMingen™), APC-CD71 (clone: CY1G4; Biolegend), APC-IgG2a (clone: MOPC-173; Biolegend), Brilliant Violet 510-CD98 (clone: UM7F8; BD OptiBuil™), and Brilliant Violet 510-IgG1 (clone: X40; BD OptiBuil™). Fixable Viability Stain 620 (BD Horizon™, USA) was used to evaluate cell viability in all experiments. The phenotypic analysis above was conducted using a BD FACS Canto II Flow Cytometer and analyzed by FlowJo 10.4 software (Ashland, OR, USA).
8
Acute Malaria PBMC Immunophenotyping
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PBMCs collected during acute malaria were used for MBC subset and plasmablast/plasma cell phenotyping. Fluorochrome-conjugated, mouse anti-human monoclonal antibodies were used to stain 1 million PBMCs/100 μL FACS buffer. A cocktail consisting of the following mouse monoclonal antibodies was used: FITC-CD19, APC-CD21, APC/fire-CD27, AF700-CD38, PE-IgM and PE/Cy7-IgD (Biolegend, San Diego, CA, USA). After staining for 15 min, cells were washed with FACS buffer. Finally, cells were suspended in 250 μL FACS buffer. The analyses were done with a flow cytometer (FACSCanto II, Becton–Dickinson Immunocytometry Systems, San Jose, CA, USA). Data were processed using FlowJo software (Tree Star, San Carlos, CA, USA).
9
Multicolor Flow Cytometry Analysis of Immune Cells
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Blood was collected by retro‐orbital puncture in heparinized micro‐hematocrit capillary tubes 4 days prior to harvest. Erythrocytes were lysed with ACK lysis buffer (155 mM ammonium chloride, 10 mM potassium bicarbonate, 0.01 mM EDTA, pH 7.4). WBC were resuspended in 3% FBS in PBS, blocked with 2 μg/ml of FcgRII/III, and then stained with a cocktail of antibodies. Monocytes were identified as CD115hi and subsets as Ly6‐Chi and Ly6‐Clo; neutrophils were identified as CD115loLy6‐ChiLy6‐Ghi. T cells were identified as CD4‐high and CD8‐high, respectively. Mature B cells were identified as B220‐high, CD19‐high, and IgM‐high cells, while B cell progenitors were identified as CD19‐high, B220‐high, and IgM‐low. Flow cytometry was performed using a BD LSRII and analyzed using FlowJo software (Tree Star). The following antibodies were used (all from BioLegend): FITC‐Ly6‐C (AL‐21), PE‐CD115 (AFS98), APC‐Ly6‐G (1A8), BV412‐CD8 (53‐6.7), APC‐cy7‐CD4 (RM4‐5), PE‐cy7‐B220 (RA3‐6B2), APC‐CD19 (6D5), and PE‐IgM (RMM‐1); and all were used at a 1:500 dilution.
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