Igm pe cy7

Manufactured by BioLegend

IgM-PE-Cy7 is a fluorescent-conjugated antibody product manufactured by BioLegend. It is designed for use in flow cytometry applications to detect and analyze IgM-positive cells.

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5 protocols using igm pe cy7

Multiparameter Analysis of Immune Cell Subsets

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Single-cell suspensions from spleens, bone marrow, or peripheral lymph nodes were blocked for 30 min using FBS. Cell-surface staining was achieved by incubating cells at 4°C for 30 min with fluorescence-conjugated anti–mouse antibodies (clone; source): XBP1s–Alexa Fluor 647 (Q3-695; BD), XBP1s-phycoerythrin (PE; Q3-695; BD), B220–Alexa Fluor 488 (RA3-6B2; BioLegend), B220-BV605 (RA3-6B2; BioLegend), CD43-PE (eBioR2/60; eBioscience), CD19–Alexa Fluor 647 (6D5; BioLegend), IgM-PE-Cy7 (RMM-1; BioLegend), IgD-FITC (11-26c.2a; BioLegend), GL7-PE (GL7; BioLegend), AA4.1-PE-Cy7 (AA4.1; BioLegend), CD1d-PerCP-Cy5.5 (1B1; BioLegend), CD23-FITC (B3B4; BioLegend), CD3-APC-Cy7 (145-2C11; BioLegend), CD4-BV605 (RM4-5; BioLegend), CD8α-PE-Cy7 (53–6.7; BioLegend), and CD138-PE (281–2; BioLegend). Viability staining was accomplished using DAPI exclusion during acquisition. Acquisition of B, T, and dendritic cell populations was performed on an LSRII cytometer (BD) harboring a custom configuration for the Wistar Institute. Cytometry data were analyzed using FlowJo software (7.6.1; Tree Star Inc.).

Tang C.H., Chang S., Paton A.W., Paton J.C., Gabrilovich D.I., Ploegh H.L., Del Valle J.R, & Hu C.C. (2018). Phosphorylation of IRE1 at S729 regulates RIDD in B cells and antibody production after immunization. The Journal of Cell Biology, 217(5), 1739-1755.

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Flow Cytometric Immunophenotyping of Immune Cells

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For flow cytometry, cells were stained with mAbs in PBS with 1% BSA for 20 minutes in the dark at 4°C. Cells were subsequently washed and fixed with a 1% paraformaldehyde/PBS solution before analysis on BD FACSCalibur flow cytometer (BD Immunocytometry Systems, [BDIS] San Jose, CA) or MACSQuant® Analyzer (Miltenyi Biotec). The anti-human antibodies used were CD19 Pacific Blue, CD24 PE-Cy7, CD38 APC, CD27 APC-Cy7, CD21 FITC, CD1c PE (all from Biolegend, San Diego, CA), CD10 PE, CD5 PE, CD23 PE, IgD PE, IgM PE-Cy5 (all from BD Bioscience), BAFF-R PE (eBioscience Inc., San Diego, CA). The following anti-mouse antibodies were used: CD24 Pacific Blue, CD21 FITC, CD23 PE, BAFF-R PE, CD21 APC, SA-APC-Cy7 (secondary antibody for IgD biotin staining), IgM PE-Cy7 (all from Biolegend), and IgD Biotin (eBioscience). Living cells were identified by forward scatter and side scatter gating and/or exclusion of 7-aminoactinomycin-D (eBioscience) added immediately prior to data acquisition or fixation. Flow cytometry data analysis was performed using Flowjo data analysis software (TreeStar, Ashland, OR).

Benitez A., Weldon A.J., Tatosyan L., Velkuru V., Lee S., Milford T.A., Francis O.L., Hsu S., Nazeri K., Casiano C.M., Schneider R., Gonzalez J., Su R.J., Baez I., Colburn K., Moldovan I, & Payne K.J. (2014). Differences in Mouse and Human Non-Memory B Cell Pools. Journal of immunology (Baltimore, Md. : 1950), 192(10), 4610-4619.

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Multicolor FACS analysis of EVs

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A FACSAria II SORP cell sorter was used to collect flow cytometry (FACS) data using FACSDiva v8.0 software (BD Biosciences; San Jose CA), at the Cold-Ocean Deep-Sea Research Facility (Memorial University of Newfoundland). Data analysis was performed using FlowJo v10.0.5 (Tree Star; Ashland, OR.). All reagents were from eBioscience (San Diego, CA) and are rat anti-mouse antibodies, unless otherwise stated. Colour compensation was performed using single-stained OneComp beads (01–1111) with all fluorophores. Cells were stimulated to produce EVs as described above using 1° Ab pre-incubated with biotinylated 2° Ab. Isotype or anti-CD24-treated cells were stained with 0.5 µg M1/69 CD24-FITC (11–0242) or Streptavidin-FITC (11–4317), respectively. All cells were stained with 1.25 µg Siglec-2-PE (126111; Biolegend; San Diego CA), 0.625 µg CD63 PerCP-eFluor710 (46–0631), 1.25 µg IgM PE-Cy7 (25–5790), 0.625 µg Siglec-G APC (17–5833), MHC-II (I-A/I-E) Alexa Fluor 700 (56–5321), and 0.625 µg Ter119 APC-eFluor780 (47–5921). Matching isotype Ab controls were used to confirm the absence of non-specific Ab binding and to set thresholds. Captured MVs were stained with the same fluorophores except with 5 µL Annexin V Alexa488 (Thermo Fisher Scientific) instead of anti-CD24-FITC or Streptavidin-FITC.

Ayre D.C., Chute I.C., Joy A.P., Barnett D.A., Hogan A.M., Grüll M.P., Peña-Castillo L., Lang A.S., Lewis S.M, & Christian S.L. (2017). CD24 induces changes to the surface receptors of B cell microvesicles with variable effects on their RNA and protein cargo. Scientific Reports, 7, 8642.

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Comprehensive B Cell Immunophenotyping

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Cells (1× 10⁶) were collected by centrifugation and washed twice in PBS/2% FCS, and the cell pellet was resuspended in 100 μl of PBS/2% FCS. Fluorophore-conjugated surface marker Abs were added at a 1:100 dilution and incubated on ice for 20 min. Cells were washed in PBS/2% FCS and resuspended in PBS/2% FCS for analysis on a BD LSR II Flow Cytometer. The Abs used in the FACS analysis were as follows: CD21-FITC (553818) BD Biosciences Rat, CD23-PE (561773) BD Pharmingen Rat, CD23-Pacific blue (101616) BioLegend Rat, IgD-allophycocyanin (405713) BioLegend Rat, IgM-PE-Cy7 (406513) BioLegend Rat, B220-Pacific blue (558108) BD Biosciences Rat, B220-PE (553089) BD Biosciences Rat, and CD22-PE (BD 553384). The LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific) was used to distinguish between live and dead cells. Data were analyzed with FlowJo software. The gating strategy was that cells were initially gated for live lymphocytes followed by analysis of B220⁺ cells.

Thomsen I., Kunowska N., de Souza R., Moody A.M., Crawford G., Wang Y.F., Khadayate S., Whilding C., Strid J., Karimi M.M., Barr A.R., Dillon N, & Sabbattini P. (2021). RUNX1 Regulates a Transcription Program That Affects the Dynamics of Cell Cycle Entry of Naive Resting B Cells. The Journal of Immunology Author Choice, 207(12), 2976-2991.

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Multiparametric Flow Cytometry Analysis

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Flow cytometry was performed on bone marrow, spleen, and thymus samples. For the latter two, cells were forced through a 40 µm nylon cell strainer in PBS with 2% fetal calf serum (FCS). For the bone marrows, cells were collected by flushing femurs and tibias with PBS containing 2% FCS. After RBC lysis, collected cells were washed and resuspended in PBS with 2% FCS and counted on a hemocytometer. For fluorescence-activated cell sorting (FACS) analyses, cells were stained for 30 mins at 4 °C with the following antibodies: CD3 FITC (BioLegend San Diego, CA; 17A2), CD4 PE (BD Biosciences; GK1.5), CD8 APC (BD Biosciences; 53–6.7), CD19 BV605 (Biolegend; 6D5), CD43 APC (Biolegend; S11), IgD e450 (eBiosciences; 11–26 Waltham, MA), IgM PE-Cy-7 (Biolegend; RMM-1), B220 PE (eBiosciences RA3–6B2), Gr-1 FITC (Biolegend RB-6–8C5), Mac1 PE (BD Biosciences; M1/70), and CD11c APC (eBiosciences; N418). FACS analysis was performed on a LSR Fortessa flow cytometer (BD Biosciences) with FACSDiva software (BD Biosciences). Data was analyzed with FlowJo software (Tree Star Inc, Ashland, OR).

Yu F.P., Dworski S, & Medin J.A. (2018). Deletion of MCP-1 Impedes Pathogenesis of Acid Ceramidase Deficiency. Scientific Reports, 8, 1808.

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