anti mouse igg3 pe cy7  (SouthernBiotech)

Journal: Science Advances

Article Title: IgM and IgG1 B cell receptors differentially affect B cell fates and dictate the pathogenesis of mature B cell lymphomas

doi: 10.1126/sciadv.adp9391

Figure Lengend Snippet: ( A ) IGHM (left) and IGHG1 (right) expression as transcripts per million (TPM) in IgM high ( n = 111) or IgG1 high ( n = 63) samples. ( B ) Kaplan-Meir curves displaying progression-free survival (probability) in IgM and IgG1 high samples. ( C ) Distribution of distinct DLBCL subtypes in IgM and IgG1 high groups. ( D to F ) Kaplan-Meir curves for progression-free survival (probability) in IgM high and IgG1 high in BN2 (D), EZB (E), and Other (F) genetic subtypes. sgRNA, single guide RNA. ( G ) Engineering of switched isogenic human lymphoma cell lines using CRISPR-Cas9. ( H ) IgM and IgG1 expression in BJAB, OCI-Ly7. and HBL-1 cells before and after CRISPR-Cas9 editing. ( I ) Tumor growth of IgM or IgG1 cells (1 million per mouse) subcutaneously injected into NSG mice ( n = 5 mice). ( J ) Tumor growth curves of IgM or IgG1 expressing BJAB subcutaneously injected into NSG mice. ( K ) Surface expression of Ig-kappa light chain (Igκ) in IgM BJAB cells (gray), IgM tumors (gray dashed), and IgG1 BJAB cells (red), tumors which grew from IgG1 BJAB cells (red dashed) and BJAB IgM cells stained with secondary Ab (no Ab control, black). ( L ) Cotransplantation of IgG1 and IgM lymphoma cells mixed at 1:1. Bottom; Representative plots of IgM and IgG1 at days 0 and 21 posttransplant. ( M ) Quantification of IgM and IgG1 cell frequency in tumors after 21 days ( n = 4 for Mino IgG1, Mino IgM, and BJAB IgG1; n = 5 for BJAB IgM). ( N and O ) Bar graphs quantifying percent IgM and IgG1 BJAB (N) and Mino (O) cells after mixing (day 0) and upon coculture for 5 or 8 days. Statistical significance calculated by log-rank test [(B) and (D) to (F)], t test (I), and two-way ANOVA [(M) to (O)]. Error bars represent ± SEM; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001. [(G) and (L)] Created in BioRender. Shukla, V. (2025) https://BioRender.com/1mfdxwl . See also fig. S1.

Article Snippet: Anti-mouse IgG3 PE/Cy7 , Southern Biotech , 1100-17.

Techniques: Expressing, CRISPR, Injection, Staining, Control

Journal: Science Advances

Article Title: IgM and IgG1 B cell receptors differentially affect B cell fates and dictate the pathogenesis of mature B cell lymphomas

doi: 10.1126/sciadv.adp9391

Figure Lengend Snippet: ( A ) Schematic of the experimental design. Cd19 cre Tet2 / Tet3- deficient DKO B cells (CD45.2+) were isolated from the spleen and cultured for 4 days using an iGB in vitro culture system. TET-deficient B cells (2 million) were transplanted into sublethally irradiated CD45.1+ recipient mice. ( B ) Representative flow cytometry plot showing the frequency of IgM and IgG1 before transplantation in TET-deficient YFP+, CD45.1− B cells 4 days after iGB culture. ( C ) Relative frequency of IgM and IgG1 expressing YFP+, CD45.2+ B cells in blood over time. IgM and IgG1 frequencies are normalized to their respective pretransplantation frequency ( n = 9 from two independent experiments). ( D ) Representative flow cytometry plot of YFP+, CD45.1− splenic B cells at week 10 posttransplantation (left). Quantification of IgM and IgG1 frequency (right) ( n = 9 from two independent experiments). ( E ) Quantification of the absolute number of IgM and IgG1 splenic B cells gated from YFP+, CD45.1− B cells. Cell numbers are normalized to IgM ( n = 9 from two independent experiments). ( F ) Representative flow cytometry plots of proliferating IgM and IgG1 cells with Ki67 staining from YFP+, CD45.1− B cells (left). Quantification of Ki67+ from YFP+, CD45.1− splenic B cells (right) ( n = 6 from two independent experiments). ( G ) Representative flow cytometry plots of apoptotic IgM and IgG1 cells measured by cleaved caspase-3 staining from YFP+, CD45.1− cells (left). Quantification of cleaved caspase-3 from YFP+, CD45.1− splenic B cells (right) ( n = 6 from two independent experiments). Statistical significance is calculated by ordinary two-way ANOVA test with Šídák multiple comparisons test (C) and paired t test [(D) to (G)]. Error bars represent means ± SEM; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001. Schematic in (A) created in BioRender. Shukla, V. (2025) https://BioRender.com/1mfdxwl . See also fig. S2.

Article Snippet: Anti-mouse IgG3 PE/Cy7 , Southern Biotech , 1100-17.

Techniques: Isolation, Cell Culture, In Vitro, Irradiation, Flow Cytometry, Transplantation Assay, Expressing, Staining

Journal: Science Advances

Article Title: IgM and IgG1 B cell receptors differentially affect B cell fates and dictate the pathogenesis of mature B cell lymphomas

doi: 10.1126/sciadv.adp9391

Figure Lengend Snippet: ( A ) Schematic for SRBC immunization. ( B ) Representative plots for gating GC B cells (left). Quantification of cleaved caspase-3 from IgM and IgG1 GC B cells (right). Connecting lines show data points within each mouse. ( C ) Schematic of iGB cocultures. ( D ) Representative plots for IgM and IgG1 frequency from CD19+CD138−, 12 hours poststimulation (BCR) with anti-kappa antibody (1 μg/ml). ( E ) Quantification of IgG1 and IgM B cell frequency with or without BCR stimulation. IgM and IgG1 are normalized to respective frequencies under untreated conditions ( n = 7, three independent experiments). ( F ) Quantification of IgM and IgG1 B cell numbers ( n = 7, three independent experiments). ( G ) Representative flow plots (left) and quantification of cleaved caspase-3 (right). Cleaved caspase-3 is normalized to IgM cells in the untreated group ( n = 17 from seven independent experiments). ( H ) Genome browser tracks of RNA-seq data in the IgH locus ( Ighg1 and Ighm genes are highlighted). Black and red tracks are from IgM and IgG1 cells from untreated and BCR-stimulated groups. Tracks are averaged from three replicates. ( I ) Volcano plot displaying DEGs in IgG1- versus IgM expressing cells from untreated (left) and BCR-stimulated conditions (right). ( J ) Up-regulated (red) and down-regulated (gray) pathways identified from DEGs in IgG1 compared with IgM using Metascape. The x axis represents the z score. ( K ) Gene set enrichment analysis (GSEA) of apoptosis and abnormal mitochondrial morphology gene sets. The y axis denotes enrichment score. NES, normalized enrichment score; P nominal P value. Statistical significance is calculated by the paired t test (B) and ordinary two-way ANOVA with Šídák multiple comparisons test [(E) and (F)]. Error bars represent ± SEM; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001. Schematics in (A) and (C) created in BioRender. Shukla, V. (2025) https://BioRender.com/1mfdxwl . See also fig. S3.

Article Snippet: Anti-mouse IgG3 PE/Cy7 , Southern Biotech , 1100-17.

Techniques: RNA Sequencing, Expressing

Journal: Science Advances

Article Title: IgM and IgG1 B cell receptors differentially affect B cell fates and dictate the pathogenesis of mature B cell lymphomas

doi: 10.1126/sciadv.adp9391

Figure Lengend Snippet: ( A ) Representative plots of IgM and IgG1 GC B cells (Fas+, CD38low) (left) 14 days postimmunization with SRBCs. Histograms show MitoTracker Green staining in IgM and IgG1 cells (middle). Median fluorescence intensity of MitoTracker Green in IgM and IgG1 cells (right) ( n = 8). ( B ) Representative ImageStream analysis of IgM and IgG1 GC B cells stained with DAPI (nucleus), Tomm20 (PE: mitochondria), IgG1 (FITC), and IgM (PE-Cy7). ( C ) Histograms showing MitoTracker Green in IgM and IgG1 cells from iGB cultures stimulated with anti-kappa (1 μg/ml) for 12 hours (left). Quantification of relative MitoTracker Green mean fluorescence intensity (MFI) (middle). Values are normalized to untreated IgM from respective experiments. Relative MitoTracker Green MFI also normalized to cell size (right) ( n = 6 from two independent experiments). ( D ) Flow cytometry histograms showing MitoTracker Green staining (top) and quantification of MitoTracker Green MFI (bottom) in IgM and IgG1 splenic B cells from heterozygous IgH γ1μ /+ mice ( n = 3). ( E ) Representative confocal microscopy images of splenic B cells from heterozygous IgH γ1μ /+ mice (left) and quantified as mean fluorescence intensity (right). ( F ) Experimental design with IgG1 cells isolated from expressing homozygous IgH γ1μ/γ1μ mice and treated with TAT-Cre for 2 hours and stimulated with anti-kappa antibody. ( G ) Representative flow plots of IgM and IgG1 staining of IgH γ1μ/γ1μ splenic B cells before TAT-Cre treatment and 24 and 48 hours after TAT-Cre treatment. ( H ) Histograms showing MitoTracker Green staining (left) and quantification of MitoTracker Green MFI (right) in IgM and IgG1 B cells from IgH γ1μ /γ1μ mice 48 hours post-BCR anti-kappa stimulation (2 μg/ml) ( n = 5). Statistical significance is calculated by the paired t test (A), ordinary two-way ANOVA with Šídák multiple comparisons test [(C), (D), and (H)], and unpaired t test (E). Error bars represent ± SEM; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001. See also fig. S4.

Article Snippet: Anti-mouse IgG3 PE/Cy7 , Southern Biotech , 1100-17.

Techniques: Staining, Fluorescence, Flow Cytometry, Confocal Microscopy, Isolation, Expressing

Journal: Science Advances

Article Title: IgM and IgG1 B cell receptors differentially affect B cell fates and dictate the pathogenesis of mature B cell lymphomas

doi: 10.1126/sciadv.adp9391

Figure Lengend Snippet: ( A ) Changes in OCR measured over time in IgM (black) and IgG1 (red) IgH γ1μ /+ B cells stimulated with anti-kappa antibody, treated with oligomycin, phenylhydrazone (FCCP), and antimycin A and rotenone. ( B ) Changes in OCR measured over time in IgM and IgG1 BJAB lymphoma cells treated with anti-Igκ antibody, oligomycin, FCCP, antimycin A, and rotenone. ( C ) Representative flow cytometry plots of IgM and IgG1 splenic GC B cells (Fas+, CD38 low) stained with MitoTracker Green ( x axis) and TMRM ( y axis). Gated population indicates dysfunctional mitochondria (left), identified as lower TMRM and positive MitoTracker Green staining. Quantification of the frequency of dysfunctional mitochondria in IgM and IgG1 GC B cells (right) ( n = 3). ( D ) Representative flow cytometry plots of IgM and IgG1 B cells from in vitro iGB cultures stained with MitoTracker Green ( x axis) and TMRM ( y axis) (left). Quantification of the frequency of dysfunctional mitochondria measured by identifying as lower TMRM and positive MitoTracker Green staining (right) ( n = 5 from two independent experiments). ( E ) Frequency of cleaved caspase-3–positive cells in IgM and IgG1 B cells from in vitro iGB cultures ( n = 5 from two independent experiments). ( F and G ) Representative flow cytometry plots of OCI-Ly7 (F) and BJAB (G) IgM and IgG1 lymphoma cells stained with MitoTracker Green and TMRM after BCR treatment for 24 hours (left). Quantification of the frequency of dysfunctional mitochondria (right) ( n = 6 from two independent experiments (F) and n = 3 (G) (right). Statistical significance is calculated using a paired t test (C) and ordinary two-way ANOVA with Šídák multiple comparisons test [(D) to (G)]. Error bars represent means ± SEM; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001. Schematic in (F) created in BioRender. Shukla, V. (2025) https://BioRender.com/1mfdxwl . See also fig. S4.

Article Snippet: Anti-mouse IgG3 PE/Cy7 , Southern Biotech , 1100-17.

Techniques: Flow Cytometry, Staining, In Vitro

Journal: Science Advances

Article Title: IgM and IgG1 B cell receptors differentially affect B cell fates and dictate the pathogenesis of mature B cell lymphomas

doi: 10.1126/sciadv.adp9391

Figure Lengend Snippet: ( A ) Calcium flux measured by fluorescence intensity of Fluo4 ( y axis) over time in IgM and IgG1 IgH γ1μ /+ splenic B cells in response to BCR (anti-Igκ) stimulation. ( B ) Calcium flux measured by fluorescence intensity of Fluo4 in IgM and IgG1 OCI-Ly7 lymphoma cells in response to BCR (anti-Igκ) stimulation. ( C ) Schematic of the iGB in vitro culture system with treatment groups. ( D ) Representative flow cytometry plots of IgM and IgG1 B cells derived from iGB in vitro cultures stained with MitoTracker Green ( x axis) and TMRM ( y axis) 12 hours posttreatment. ( E ) Quantification of dysfunctional mitochondria frequency in IgM and IgG1 B cells from iGB in vitro cultures ( n = 6 from two independent experiments). ( F ) Representative flow cytometry plots of cleaved caspase-3 from IgM and IgG1 B cells from iGB in vitro cultures 12 hours posttreatment. ( G ) Quantification of relative frequency of cleaved caspase 3 from IgM and IgG1 B cells from iGB in vitro cultures 12 hours poststimulation. Values are normalized to DMSO IgM and IgG1. ( H ) Proposed model showing that IgG1 B cell signaling triggers greater calcium flux, leading to increased mitochondrial dysfunction, which contributes to reduced cell survival. Dampening of calcium flux with BAPTA-AM rescues the survival of IgG1 expressing B cells. Statistical significance is calculated by repeated measures two-way ANOVA with Tukey’s multiple comparisons test [(E) and (G)]. Error bars represent means ± SEM; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001. Schematics in (C) and (H) created in BioRender. Shukla, V. (2025) https://BioRender.com/1mfdxwl . See also fig. S5.

Article Snippet: Anti-mouse IgG3 PE/Cy7 , Southern Biotech , 1100-17.

Techniques: Fluorescence, In Vitro, Flow Cytometry, Derivative Assay, Staining, Expressing

Journal: Cell Death and Differentiation

Article Title: MyD88 is an essential component of retinoic acid-induced differentiation in human pluripotent embryonal carcinoma cells

doi: 10.1038/cdd.2017.124

Figure Lengend Snippet: Inhibition of MyD88 facilitates RA-induced differentiation of 2102Ep cells. 2102Ep cells were treated with MyD88 PepInh for 24 h and then with MyD88 PepInh+RA (refreshed daily) for an additional 11 days. Cells from three independent replicates were assessed via flow cytometry (a) for differentiation status using pluripotency marker SSEA4. (a) Open lines represent negative staining controls for each sample. Filled lines represent the stained sample. Green represents the SSEA4Neg cells and red the SSEA4Pos cells, as determined by internal negative staining controls. 2102Ep cells were shown to be almost completely SSEA4Pos in –RA conditions (a–i) and 62.00±11.86% SSEA4Neg when force differentiated using a siRNA knockdown of Sox2 protocol we have previously described (a–ii),45 differences that were found to be statistically significant (b). Cells grown in control PepInh –RA (a–iii), MyD88 PepInh –RA (a–iv) and control PepInh +RA (a–v) conditions were found to remain almost completely SSEA4Pos (a–iii–v: c–d, statistical analysis). In contrast, cells grown in MyD88 PepInh+RA conditions for 12 days were found to be ~1:1 SSEA4Pos:SSEA4Neg (a–vi: d, statistical analysis). Although this was initially thought to suggest the presence of two sub-populations, additional experiments (Supplementary Data 4) suggest that this is owing to a small population of cells that persist through RA treatment, which can proliferate to a sizable populations over this time-scale. SSEA4Pos and SSEA4Neg populations (a–vi) were isolated via FACS and shown by qPCR to express Oct4-Sox2-Nanog at high and low levels respectively (e). Collectively, this indicates a model (f) where 2102Ep cells can be transitioned to a stable, apparently primed self-renewal (SRPR) state when MyD88 is consistently inhibited, a state that can be differentiated by RA. *P-value< 0.05; **P-value <0.01

Article Snippet: Flow cytometry/FACS For each sample, after dissociation using EDTA (1 mM), 1 million cells were assayed with PE-conjugated mouse IgG3 anti-SSEA4 antibody (MC-813-70, R&D Systems), using PE-conjugated mouse IgG3 (133316, R&D Systems) and unstained samples as isotype and autoflourescence, respectively.

Techniques: Inhibition, Flow Cytometry, Marker, Negative Staining, Staining, Knockdown, Control, Isolation

Journal: Cell Death and Differentiation

Article Title: MyD88 is an essential component of retinoic acid-induced differentiation in human pluripotent embryonal carcinoma cells

doi: 10.1038/cdd.2017.124

Figure Lengend Snippet: A potential role for MyD88 in mesodermal differentiation of 2102Ep cells. 2102Ep cells were treated with specific endoderm, mesoderm and ectoderm differentiation kits to assess whether the MyD88 mechanism was RA-specific. Morphology images show 2102Ep cells stimulated to differentiate down endoderm (a, b), ectoderm (c, d) or mesoderm (e, f) lineages using specific differentiation kits following treatment with (d–f) or without (a–c) MyD88 PepInh. Differentiation was only apparent in cells treated with the MyD88 PepInh and then a mesodermal differentiation protocol (f). Cells were subsequently assessed for differentiation status using SSEA4 flow cytometry and Oct4-Sox2-Nanog qPCR expression levels (g–m). (g) Open lines represent negative staining controls for each sample. Filled lines represent the stained sample. Green represents the SSEA4Neg cells and red the SSEA4Pos cells, as determined by the internal negative staining controls (i: untreated control, ii: mesoderm kit only, iii: MyD88 PepInh+mesoderm kit, iv: ectoderm kit only, v: MyD88 PepInh+ectoderm kit, vi: endoderm kit only, vii: MyD88 PepInh+endoderm kit). This analysis indicated that 2102Ep cells treated with MyD88 PepInh+mesoderm protocol produce a population of SSEA4Neg cells (a-iii: 38.51±2.1%) that was significantly larger (h) than observed in control cells (a-ii: 15.54±1.15%). Post-FACS isolation qPCR analysis of Oct4-Sox2-Nanog confirmed that these were differentiating cells (i). Cells treated with MyD88+ectoderm protocol produced a significant (a-iv: 3.66±0.13% a-v: 13.63±0.08% j statistical analysis, k: qPCR) SSEA4Neg population that was insufficient in size to isolate by FACS for qPCR analysis (k). Treatment of cells with the endoderm protocol did not result in differentiation (a-vi: 2.08 ±0.08% a-vii: 5.43 ±0.07% l: statistical analysis, m: qPCR). Morphology data indicate a potential role for MyD88 in mesodermal differentiation. *P-value< 0.05

Article Snippet: Flow cytometry/FACS For each sample, after dissociation using EDTA (1 mM), 1 million cells were assayed with PE-conjugated mouse IgG3 anti-SSEA4 antibody (MC-813-70, R&D Systems), using PE-conjugated mouse IgG3 (133316, R&D Systems) and unstained samples as isotype and autoflourescence, respectively.

Techniques: Flow Cytometry, Expressing, Negative Staining, Staining, Control, Isolation, Produced

Journal: Cell Death and Differentiation

Article Title: MyD88 is an essential component of retinoic acid-induced differentiation in human pluripotent embryonal carcinoma cells

doi: 10.1038/cdd.2017.124

Figure Lengend Snippet: Differentiated NTera2 cells secrete specific protein profiles sufficient to promote differentiation. Undifferentiated NTera2 cells were grown for 7 days in media conditioned by (collected for 7 or 14 days) undifferentiated (UndiffCon) or pre-differentiated (DiffCon) NTera2 cells, after which SSEA4 expression levels were assessed using flow cytometry (a). Open lines represent negative staining controls for each sample. Filled lines represent the stained sample. Green represents the SSEA4Neg cells and red the SSEA4Pos cells, as determined by internal negative staining controls (a–i: −RA & a–ii: +RA controls). Treatment with DiffCon media (collected for 7 (v) or 14 (vi) days) was found to reduce SSEA4 expression levels, data that were found to be statistically significant (b). This was accompanied by statistically significant decreases in qPCR measured levels of Oct4-Sox2-Nanog expression (c, 7 day DiffCon). In contrast, UndiffCon media (collected for 7 (iii) or 14 (iv) days) had no effect on SSEA4 expression (a, b). No statistically significant changes in qPCR measured levels of Oct4-Sox2-Nanog expression were observed for UndiffCon media (c).These data indicate that differentiated cells secrete factors that promote differentiation. *P-value< 0.05; **P-value <0.01, ns= not significant

Article Snippet: Flow cytometry/FACS For each sample, after dissociation using EDTA (1 mM), 1 million cells were assayed with PE-conjugated mouse IgG3 anti-SSEA4 antibody (MC-813-70, R&D Systems), using PE-conjugated mouse IgG3 (133316, R&D Systems) and unstained samples as isotype and autoflourescence, respectively.

Techniques: Expressing, Flow Cytometry, Negative Staining, Staining

Journal: Cell Death and Differentiation

Article Title: MyD88 is an essential component of retinoic acid-induced differentiation in human pluripotent embryonal carcinoma cells

doi: 10.1038/cdd.2017.124

Figure Lengend Snippet: Selected genes expressed by 2102Ep cells treated with MyD88 compared with control PepInh, control PepInh compared with control PepInh+RA, and MyD88 PepInh compared with SSEA4 Neg cells

Article Snippet: Flow cytometry/FACS For each sample, after dissociation using EDTA (1 mM), 1 million cells were assayed with PE-conjugated mouse IgG3 anti-SSEA4 antibody (MC-813-70, R&D Systems), using PE-conjugated mouse IgG3 (133316, R&D Systems) and unstained samples as isotype and autoflourescence, respectively.

Techniques: Control