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Becton Dickinson facsaria iii cell sorter
Repression of activation-induced cytidine deaminase (AID) induction in germinal center B cells (GCBs) from Pten fl/fl Cγ1 Cre/+ mice. (A) Flow cytometry sorting of in vitro induced IgM-BCR expressing splenic B cells from Pten +/+ Cγ1 Cre/+ and Pten fl/fl Cγ1 Cre/+ mice. Pure splenic B cells from control and KO mice (three mice for each group) were stimulated with LPS plus interleukin-4 (IL-4) for 4 days before sorting. (B) The expression levels of PTEN, p-AKT, p-FoxO1/3a, and AID were determined in Pten +/+ Cγ1 Cre/+ and Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells (left). Reduced PTEN, AID expression and enhanced p-AKT and p-FoxO1/3a expression were observed in Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells. The in vitro induced IgM-BCR expressing B cells were isolated by the <t>FACSAria</t> <t>III</t> Cell Sorter as described in (A) . Statistical comparison of protein expression level was also shown in right. Ratio values of different proteins from Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells were normalized to that of the Pten +/+ Cγ1 Cre/+ IgM-BCR expressing B cells. Data were given from one representative of at least three independent experiments. * p
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1) Product Images from "PTEN-Regulated AID Transcription in Germinal Center B Cells Is Essential for the Class-Switch Recombination and IgG Antibody Responses"

Article Title: PTEN-Regulated AID Transcription in Germinal Center B Cells Is Essential for the Class-Switch Recombination and IgG Antibody Responses

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.00371

Repression of activation-induced cytidine deaminase (AID) induction in germinal center B cells (GCBs) from Pten fl/fl Cγ1 Cre/+ mice. (A) Flow cytometry sorting of in vitro induced IgM-BCR expressing splenic B cells from Pten +/+ Cγ1 Cre/+ and Pten fl/fl Cγ1 Cre/+ mice. Pure splenic B cells from control and KO mice (three mice for each group) were stimulated with LPS plus interleukin-4 (IL-4) for 4 days before sorting. (B) The expression levels of PTEN, p-AKT, p-FoxO1/3a, and AID were determined in Pten +/+ Cγ1 Cre/+ and Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells (left). Reduced PTEN, AID expression and enhanced p-AKT and p-FoxO1/3a expression were observed in Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells. The in vitro induced IgM-BCR expressing B cells were isolated by the FACSAria III Cell Sorter as described in (A) . Statistical comparison of protein expression level was also shown in right. Ratio values of different proteins from Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells were normalized to that of the Pten +/+ Cγ1 Cre/+ IgM-BCR expressing B cells. Data were given from one representative of at least three independent experiments. * p
Figure Legend Snippet: Repression of activation-induced cytidine deaminase (AID) induction in germinal center B cells (GCBs) from Pten fl/fl Cγ1 Cre/+ mice. (A) Flow cytometry sorting of in vitro induced IgM-BCR expressing splenic B cells from Pten +/+ Cγ1 Cre/+ and Pten fl/fl Cγ1 Cre/+ mice. Pure splenic B cells from control and KO mice (three mice for each group) were stimulated with LPS plus interleukin-4 (IL-4) for 4 days before sorting. (B) The expression levels of PTEN, p-AKT, p-FoxO1/3a, and AID were determined in Pten +/+ Cγ1 Cre/+ and Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells (left). Reduced PTEN, AID expression and enhanced p-AKT and p-FoxO1/3a expression were observed in Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells. The in vitro induced IgM-BCR expressing B cells were isolated by the FACSAria III Cell Sorter as described in (A) . Statistical comparison of protein expression level was also shown in right. Ratio values of different proteins from Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells were normalized to that of the Pten +/+ Cγ1 Cre/+ IgM-BCR expressing B cells. Data were given from one representative of at least three independent experiments. * p

Techniques Used: Activation Assay, Mouse Assay, Flow Cytometry, Cytometry, In Vitro, Expressing, Isolation

2) Product Images from "PTEN-Regulated AID Transcription in Germinal Center B Cells Is Essential for the Class-Switch Recombination and IgG Antibody Responses"

Article Title: PTEN-Regulated AID Transcription in Germinal Center B Cells Is Essential for the Class-Switch Recombination and IgG Antibody Responses

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.00371

Repression of activation-induced cytidine deaminase (AID) induction in germinal center B cells (GCBs) from Pten fl/fl Cγ1 Cre/+ mice. (A) Flow cytometry sorting of in vitro induced IgM-BCR expressing splenic B cells from Pten +/+ Cγ1 Cre/+ and Pten fl/fl Cγ1 Cre/+ mice. Pure splenic B cells from control and KO mice (three mice for each group) were stimulated with LPS plus interleukin-4 (IL-4) for 4 days before sorting. (B) The expression levels of PTEN, p-AKT, p-FoxO1/3a, and AID were determined in Pten +/+ Cγ1 Cre/+ and Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells (left). Reduced PTEN, AID expression and enhanced p-AKT and p-FoxO1/3a expression were observed in Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells. The in vitro induced IgM-BCR expressing B cells were isolated by the FACSAria III Cell Sorter as described in (A) . Statistical comparison of protein expression level was also shown in right. Ratio values of different proteins from Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells were normalized to that of the Pten +/+ Cγ1 Cre/+ IgM-BCR expressing B cells. Data were given from one representative of at least three independent experiments. * p
Figure Legend Snippet: Repression of activation-induced cytidine deaminase (AID) induction in germinal center B cells (GCBs) from Pten fl/fl Cγ1 Cre/+ mice. (A) Flow cytometry sorting of in vitro induced IgM-BCR expressing splenic B cells from Pten +/+ Cγ1 Cre/+ and Pten fl/fl Cγ1 Cre/+ mice. Pure splenic B cells from control and KO mice (three mice for each group) were stimulated with LPS plus interleukin-4 (IL-4) for 4 days before sorting. (B) The expression levels of PTEN, p-AKT, p-FoxO1/3a, and AID were determined in Pten +/+ Cγ1 Cre/+ and Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells (left). Reduced PTEN, AID expression and enhanced p-AKT and p-FoxO1/3a expression were observed in Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells. The in vitro induced IgM-BCR expressing B cells were isolated by the FACSAria III Cell Sorter as described in (A) . Statistical comparison of protein expression level was also shown in right. Ratio values of different proteins from Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells were normalized to that of the Pten +/+ Cγ1 Cre/+ IgM-BCR expressing B cells. Data were given from one representative of at least three independent experiments. * p

Techniques Used: Activation Assay, Mouse Assay, Flow Cytometry, Cytometry, In Vitro, Expressing, Isolation

3) Product Images from "PTEN-Regulated AID Transcription in Germinal Center B Cells Is Essential for the Class-Switch Recombination and IgG Antibody Responses"

Article Title: PTEN-Regulated AID Transcription in Germinal Center B Cells Is Essential for the Class-Switch Recombination and IgG Antibody Responses

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.00371

Repression of activation-induced cytidine deaminase (AID) induction in germinal center B cells (GCBs) from Pten fl/fl Cγ1 Cre/+ mice. (A) Flow cytometry sorting of in vitro induced IgM-BCR expressing splenic B cells from Pten +/+ Cγ1 Cre/+ and Pten fl/fl Cγ1 Cre/+ mice. Pure splenic B cells from control and KO mice (three mice for each group) were stimulated with LPS plus interleukin-4 (IL-4) for 4 days before sorting. (B) The expression levels of PTEN, p-AKT, p-FoxO1/3a, and AID were determined in Pten +/+ Cγ1 Cre/+ and Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells (left). Reduced PTEN, AID expression and enhanced p-AKT and p-FoxO1/3a expression were observed in Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells. The in vitro induced IgM-BCR expressing B cells were isolated by the FACSAria III Cell Sorter as described in (A) . Statistical comparison of protein expression level was also shown in right. Ratio values of different proteins from Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells were normalized to that of the Pten +/+ Cγ1 Cre/+ IgM-BCR expressing B cells. Data were given from one representative of at least three independent experiments. * p
Figure Legend Snippet: Repression of activation-induced cytidine deaminase (AID) induction in germinal center B cells (GCBs) from Pten fl/fl Cγ1 Cre/+ mice. (A) Flow cytometry sorting of in vitro induced IgM-BCR expressing splenic B cells from Pten +/+ Cγ1 Cre/+ and Pten fl/fl Cγ1 Cre/+ mice. Pure splenic B cells from control and KO mice (three mice for each group) were stimulated with LPS plus interleukin-4 (IL-4) for 4 days before sorting. (B) The expression levels of PTEN, p-AKT, p-FoxO1/3a, and AID were determined in Pten +/+ Cγ1 Cre/+ and Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells (left). Reduced PTEN, AID expression and enhanced p-AKT and p-FoxO1/3a expression were observed in Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells. The in vitro induced IgM-BCR expressing B cells were isolated by the FACSAria III Cell Sorter as described in (A) . Statistical comparison of protein expression level was also shown in right. Ratio values of different proteins from Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells were normalized to that of the Pten +/+ Cγ1 Cre/+ IgM-BCR expressing B cells. Data were given from one representative of at least three independent experiments. * p

Techniques Used: Activation Assay, Mouse Assay, Flow Cytometry, Cytometry, In Vitro, Expressing, Isolation

4) Product Images from "Serological Evaluation of Immunity to the Varicella-Zoster Virus Based on a Novel Competitive Enzyme-Linked Immunosorbent Assay"

Article Title: Serological Evaluation of Immunity to the Varicella-Zoster Virus Based on a Novel Competitive Enzyme-Linked Immunosorbent Assay

Journal: Scientific Reports

doi: 10.1038/srep20577

ORF9 is a membrane-associated tegument protein and interacts with gE, gB and gC. ( A ) ARPE-19 cells were infected with v-Oka, and cell-free viruses were purified by sucrose density gradient centrifugation. Purified virions were dialyzed to PBS and then treated with trypsin (0.2 mg/ml) in either the presence or absence of 1% Triton X-100 for 25 min at room temperature. Equivalent amounts of virion lysates were analysed for the indicated viral proteins in Western blotting. ( B ) v-Oka-infected ARPE-19 and uninfected ARPE-19 cells were incubated with 8H6-FITC at 37 °C or 4 °C for 30 min. v-Oka-infected ARPE-19 cells incubated with PBS at 4 °C for 30 min were used as the controls. All the samples (approximately 5,000 cells per test) were analysed using a FACSAria III cell sorter (BD Biosciences, San Jose, California, USA), and v-Oka-infected ARPE-19 cells incubated with 8H6-FITC showed a significant shift to higher fluorescence intensities. ( C ) Coimmunoprecipitation (Co-IP) of ORF9 with VZV membrane proteins in vitro . Uninfected and v-Oka-infected ARPE-19 cells were harvested in 1% Triton X-100 lysis buffer. Cell lysates (precleared with protein A beads) were incubated with 8H6 and then incubated with protein A beads. After fully washing with PBST (0.05% Tween 20 in PBS), the bound proteins were detected with VZV membrane protein mAbs by Western blot. Compared with uninfected cell lysates (lane 3), specific bands were detected by gE mAb (1B11), gC mAb (2A9) and gB mAb (11E5). The reaction of these mAbs with v-Oka-infected cell lysate was verified (lane 1). Furthermore, gE mAb (4A2), gC mAb (2D1) and gB mAb (4C8) were used to pull down ORF9, and 8H6 was used detection in the Western blot. In contrast to the uninfected cell lysate (lane 5), ORF9 was pulled down by these mAbs (lane 4).
Figure Legend Snippet: ORF9 is a membrane-associated tegument protein and interacts with gE, gB and gC. ( A ) ARPE-19 cells were infected with v-Oka, and cell-free viruses were purified by sucrose density gradient centrifugation. Purified virions were dialyzed to PBS and then treated with trypsin (0.2 mg/ml) in either the presence or absence of 1% Triton X-100 for 25 min at room temperature. Equivalent amounts of virion lysates were analysed for the indicated viral proteins in Western blotting. ( B ) v-Oka-infected ARPE-19 and uninfected ARPE-19 cells were incubated with 8H6-FITC at 37 °C or 4 °C for 30 min. v-Oka-infected ARPE-19 cells incubated with PBS at 4 °C for 30 min were used as the controls. All the samples (approximately 5,000 cells per test) were analysed using a FACSAria III cell sorter (BD Biosciences, San Jose, California, USA), and v-Oka-infected ARPE-19 cells incubated with 8H6-FITC showed a significant shift to higher fluorescence intensities. ( C ) Coimmunoprecipitation (Co-IP) of ORF9 with VZV membrane proteins in vitro . Uninfected and v-Oka-infected ARPE-19 cells were harvested in 1% Triton X-100 lysis buffer. Cell lysates (precleared with protein A beads) were incubated with 8H6 and then incubated with protein A beads. After fully washing with PBST (0.05% Tween 20 in PBS), the bound proteins were detected with VZV membrane protein mAbs by Western blot. Compared with uninfected cell lysates (lane 3), specific bands were detected by gE mAb (1B11), gC mAb (2A9) and gB mAb (11E5). The reaction of these mAbs with v-Oka-infected cell lysate was verified (lane 1). Furthermore, gE mAb (4A2), gC mAb (2D1) and gB mAb (4C8) were used to pull down ORF9, and 8H6 was used detection in the Western blot. In contrast to the uninfected cell lysate (lane 5), ORF9 was pulled down by these mAbs (lane 4).

Techniques Used: Infection, Purification, Gradient Centrifugation, Western Blot, Incubation, Fluorescence, Co-Immunoprecipitation Assay, In Vitro, Lysis

5) Product Images from "Single Amino Acid Substitution N659D in HIV-2 Envelope Glycoprotein (Env) Impairs Viral Release and Hampers BST-2 Antagonism"

Article Title: Single Amino Acid Substitution N659D in HIV-2 Envelope Glycoprotein (Env) Impairs Viral Release and Hampers BST-2 Antagonism

Journal: Viruses

doi: 10.3390/v8100285

HEK293T cells co-expressing the fusion proteins in the following combinations were analyzed with the configured BD FACSAria™ III cell sorter. ( A ) BST-2 fused Clover and HIV-2 Env WT fused to mRuby2; ( B ) BST-2 fused Clover and HIV-2 Env 1–749 fused mRuby2; ( C ) BST-2 fused Clover and HIV-2 Env N659D fused mRuby2 and ( D ) BST-2 fused Clover and HIV-2 Nef fused mRuby2. Numbers indicated in gate P4 give the percentage of FRET-positive cells included in P2 according to the previous fluorescence-activated cell sorting (FACS) configuration for the different co-expression in living cells ( Figure 6 ); ( E ) Bar diagram summarizing the FRET-positive signals from four independent experiments ( n = 4). Statistical tests give a significance with a p
Figure Legend Snippet: HEK293T cells co-expressing the fusion proteins in the following combinations were analyzed with the configured BD FACSAria™ III cell sorter. ( A ) BST-2 fused Clover and HIV-2 Env WT fused to mRuby2; ( B ) BST-2 fused Clover and HIV-2 Env 1–749 fused mRuby2; ( C ) BST-2 fused Clover and HIV-2 Env N659D fused mRuby2 and ( D ) BST-2 fused Clover and HIV-2 Nef fused mRuby2. Numbers indicated in gate P4 give the percentage of FRET-positive cells included in P2 according to the previous fluorescence-activated cell sorting (FACS) configuration for the different co-expression in living cells ( Figure 6 ); ( E ) Bar diagram summarizing the FRET-positive signals from four independent experiments ( n = 4). Statistical tests give a significance with a p

Techniques Used: Expressing, Fluorescence, FACS

BD FACSAria™ III cell sorter configuration and experimental setup of Förster resonance energy transfer (FRET)-measurements. Firstly, gate P1 selecting living cells (according to forward and sideward scatter FSC-A/SSC-A) and gate P2 selecting cells in P1 (according to forward and sideward scatter FSC-W/SSC-W in order to exclude joined or grouped cells), have been applied (not shown in this figure). Secondly, HEK293T cells expressing Clover or mRuby2 individually, in combination or as a fusion protein, were analyzed with three different filters in order to construct analysis gates and to define the FRET-positive signal. Clover (green fluorescent protein variant) was excited with the laser 488 nm and the FITC filter was used to examine fluorescence emission. mRuby2 (red fluorescent protein variant) was excited with the laser 561 nm and the PE-mCherry filter was used (panel A ). Importantly, mRuby2 and Clover showed some emission in the FRET-channel (PerCP-Cy 5.5 filter). Thus, a gate (P3) was constructed to exclude cells that emitted a false-positive signal in the FRET-channel (panel B ). As described in this image, cells co-transfected with Clover and mRuby2 exerted an aleatory FRET signal that should also be excluded. Therefore, an analysis gate (P4, in red) was applied to determine the FRET-positive cells when the FRET-adapted filters were selected (PerCP-Cy 5.5 and FITC filters, panel C ). This P4 gate excluded cells that were co-transfected with Clover and mRuby2 and thus are FRET-negative (0.5% of P2), while including cells that showed a FRET-positive signal (Clover fused mRuby2; 51.8% of P2). This gating strategy allowed for assessment the enhanced emission of the acceptor fluorochrome and therefore demonstrated the energy transfer between the two fluorochromes; namely the protein interactions.
Figure Legend Snippet: BD FACSAria™ III cell sorter configuration and experimental setup of Förster resonance energy transfer (FRET)-measurements. Firstly, gate P1 selecting living cells (according to forward and sideward scatter FSC-A/SSC-A) and gate P2 selecting cells in P1 (according to forward and sideward scatter FSC-W/SSC-W in order to exclude joined or grouped cells), have been applied (not shown in this figure). Secondly, HEK293T cells expressing Clover or mRuby2 individually, in combination or as a fusion protein, were analyzed with three different filters in order to construct analysis gates and to define the FRET-positive signal. Clover (green fluorescent protein variant) was excited with the laser 488 nm and the FITC filter was used to examine fluorescence emission. mRuby2 (red fluorescent protein variant) was excited with the laser 561 nm and the PE-mCherry filter was used (panel A ). Importantly, mRuby2 and Clover showed some emission in the FRET-channel (PerCP-Cy 5.5 filter). Thus, a gate (P3) was constructed to exclude cells that emitted a false-positive signal in the FRET-channel (panel B ). As described in this image, cells co-transfected with Clover and mRuby2 exerted an aleatory FRET signal that should also be excluded. Therefore, an analysis gate (P4, in red) was applied to determine the FRET-positive cells when the FRET-adapted filters were selected (PerCP-Cy 5.5 and FITC filters, panel C ). This P4 gate excluded cells that were co-transfected with Clover and mRuby2 and thus are FRET-negative (0.5% of P2), while including cells that showed a FRET-positive signal (Clover fused mRuby2; 51.8% of P2). This gating strategy allowed for assessment the enhanced emission of the acceptor fluorochrome and therefore demonstrated the energy transfer between the two fluorochromes; namely the protein interactions.

Techniques Used: Förster Resonance Energy Transfer, Expressing, Construct, Variant Assay, Fluorescence, Transfection

6) Product Images from "IFN-? R? Is a Key Determinant of CD8+ T Cell-Mediated Tumor Elimination or Tumor Escape and Relapse in FVB Mouse"

Article Title: IFN-? R? Is a Key Determinant of CD8+ T Cell-Mediated Tumor Elimination or Tumor Escape and Relapse in FVB Mouse

Journal: PLoS ONE

doi: 10.1371/journal.pone.0082544

The CD44+CD24- stem-like population and CD44+CD24+ population of WT MMC respond similarly to IFN-γ. WT MMC tumor cells were cultured with or without IFN-γ (50 ng/ml/10 6 cells) for 3 days and CD44+CD24- as CD44+CD24- populations were analyzed for viability (Annexin V-/PI-) and proliferation (BrdU) by flow cytometry (A), these two population where sorted from WT MMC cells using a BD FACSAria III cell sorter and cultured for 60 days, after that analysis of CD24, CD44, Sca1 and neu was done (B) by flow cytometry. Also analysis of relapsed ANV tumor is shown. Data represent two independent experiments.
Figure Legend Snippet: The CD44+CD24- stem-like population and CD44+CD24+ population of WT MMC respond similarly to IFN-γ. WT MMC tumor cells were cultured with or without IFN-γ (50 ng/ml/10 6 cells) for 3 days and CD44+CD24- as CD44+CD24- populations were analyzed for viability (Annexin V-/PI-) and proliferation (BrdU) by flow cytometry (A), these two population where sorted from WT MMC cells using a BD FACSAria III cell sorter and cultured for 60 days, after that analysis of CD24, CD44, Sca1 and neu was done (B) by flow cytometry. Also analysis of relapsed ANV tumor is shown. Data represent two independent experiments.

Techniques Used: Cell Culture, Flow Cytometry, Cytometry

7) Product Images from "PTEN-Regulated AID Transcription in Germinal Center B Cells Is Essential for the Class-Switch Recombination and IgG Antibody Responses"

Article Title: PTEN-Regulated AID Transcription in Germinal Center B Cells Is Essential for the Class-Switch Recombination and IgG Antibody Responses

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.00371

Repression of activation-induced cytidine deaminase (AID) induction in germinal center B cells (GCBs) from Pten fl/fl Cγ1 Cre/+ mice. (A) Flow cytometry sorting of in vitro induced IgM-BCR expressing splenic B cells from Pten +/+ Cγ1 Cre/+ and Pten fl/fl Cγ1 Cre/+ mice. Pure splenic B cells from control and KO mice (three mice for each group) were stimulated with LPS plus interleukin-4 (IL-4) for 4 days before sorting. (B) The expression levels of PTEN, p-AKT, p-FoxO1/3a, and AID were determined in Pten +/+ Cγ1 Cre/+ and Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells (left). Reduced PTEN, AID expression and enhanced p-AKT and p-FoxO1/3a expression were observed in Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells. The in vitro induced IgM-BCR expressing B cells were isolated by the FACSAria III Cell Sorter as described in (A) . Statistical comparison of protein expression level was also shown in right. Ratio values of different proteins from Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells were normalized to that of the Pten +/+ Cγ1 Cre/+ IgM-BCR expressing B cells. Data were given from one representative of at least three independent experiments. * p
Figure Legend Snippet: Repression of activation-induced cytidine deaminase (AID) induction in germinal center B cells (GCBs) from Pten fl/fl Cγ1 Cre/+ mice. (A) Flow cytometry sorting of in vitro induced IgM-BCR expressing splenic B cells from Pten +/+ Cγ1 Cre/+ and Pten fl/fl Cγ1 Cre/+ mice. Pure splenic B cells from control and KO mice (three mice for each group) were stimulated with LPS plus interleukin-4 (IL-4) for 4 days before sorting. (B) The expression levels of PTEN, p-AKT, p-FoxO1/3a, and AID were determined in Pten +/+ Cγ1 Cre/+ and Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells (left). Reduced PTEN, AID expression and enhanced p-AKT and p-FoxO1/3a expression were observed in Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells. The in vitro induced IgM-BCR expressing B cells were isolated by the FACSAria III Cell Sorter as described in (A) . Statistical comparison of protein expression level was also shown in right. Ratio values of different proteins from Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells were normalized to that of the Pten +/+ Cγ1 Cre/+ IgM-BCR expressing B cells. Data were given from one representative of at least three independent experiments. * p

Techniques Used: Activation Assay, Mouse Assay, Flow Cytometry, Cytometry, In Vitro, Expressing, Isolation

8) Product Images from "PTEN-Regulated AID Transcription in Germinal Center B Cells Is Essential for the Class-Switch Recombination and IgG Antibody Responses"

Article Title: PTEN-Regulated AID Transcription in Germinal Center B Cells Is Essential for the Class-Switch Recombination and IgG Antibody Responses

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.00371

Repression of activation-induced cytidine deaminase (AID) induction in germinal center B cells (GCBs) from Pten fl/fl Cγ1 Cre/+ mice. (A) Flow cytometry sorting of in vitro induced IgM-BCR expressing splenic B cells from Pten +/+ Cγ1 Cre/+ and Pten fl/fl Cγ1 Cre/+ mice. Pure splenic B cells from control and KO mice (three mice for each group) were stimulated with LPS plus interleukin-4 (IL-4) for 4 days before sorting. (B) The expression levels of PTEN, p-AKT, p-FoxO1/3a, and AID were determined in Pten +/+ Cγ1 Cre/+ and Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells (left). Reduced PTEN, AID expression and enhanced p-AKT and p-FoxO1/3a expression were observed in Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells. The in vitro induced IgM-BCR expressing B cells were isolated by the FACSAria III Cell Sorter as described in (A) . Statistical comparison of protein expression level was also shown in right. Ratio values of different proteins from Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells were normalized to that of the Pten +/+ Cγ1 Cre/+ IgM-BCR expressing B cells. Data were given from one representative of at least three independent experiments. * p
Figure Legend Snippet: Repression of activation-induced cytidine deaminase (AID) induction in germinal center B cells (GCBs) from Pten fl/fl Cγ1 Cre/+ mice. (A) Flow cytometry sorting of in vitro induced IgM-BCR expressing splenic B cells from Pten +/+ Cγ1 Cre/+ and Pten fl/fl Cγ1 Cre/+ mice. Pure splenic B cells from control and KO mice (three mice for each group) were stimulated with LPS plus interleukin-4 (IL-4) for 4 days before sorting. (B) The expression levels of PTEN, p-AKT, p-FoxO1/3a, and AID were determined in Pten +/+ Cγ1 Cre/+ and Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells (left). Reduced PTEN, AID expression and enhanced p-AKT and p-FoxO1/3a expression were observed in Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells. The in vitro induced IgM-BCR expressing B cells were isolated by the FACSAria III Cell Sorter as described in (A) . Statistical comparison of protein expression level was also shown in right. Ratio values of different proteins from Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells were normalized to that of the Pten +/+ Cγ1 Cre/+ IgM-BCR expressing B cells. Data were given from one representative of at least three independent experiments. * p

Techniques Used: Activation Assay, Mouse Assay, Flow Cytometry, Cytometry, In Vitro, Expressing, Isolation

9) Product Images from "Single Amino Acid Substitution N659D in HIV-2 Envelope Glycoprotein (Env) Impairs Viral Release and Hampers BST-2 Antagonism"

Article Title: Single Amino Acid Substitution N659D in HIV-2 Envelope Glycoprotein (Env) Impairs Viral Release and Hampers BST-2 Antagonism

Journal: Viruses

doi: 10.3390/v8100285

HEK293T cells co-expressing the fusion proteins in the following combinations were analyzed with the configured BD FACSAria™ III cell sorter. ( A ) BST-2 fused Clover and HIV-2 Env WT fused to mRuby2; ( B ) BST-2 fused Clover and HIV-2 Env 1–749 fused mRuby2; ( C ) BST-2 fused Clover and HIV-2 Env N659D fused mRuby2 and ( D ) BST-2 fused Clover and HIV-2 Nef fused mRuby2. Numbers indicated in gate P4 give the percentage of FRET-positive cells included in P2 according to the previous fluorescence-activated cell sorting (FACS) configuration for the different co-expression in living cells ( Figure 6 ); ( E ) Bar diagram summarizing the FRET-positive signals from four independent experiments ( n = 4). Statistical tests give a significance with a p
Figure Legend Snippet: HEK293T cells co-expressing the fusion proteins in the following combinations were analyzed with the configured BD FACSAria™ III cell sorter. ( A ) BST-2 fused Clover and HIV-2 Env WT fused to mRuby2; ( B ) BST-2 fused Clover and HIV-2 Env 1–749 fused mRuby2; ( C ) BST-2 fused Clover and HIV-2 Env N659D fused mRuby2 and ( D ) BST-2 fused Clover and HIV-2 Nef fused mRuby2. Numbers indicated in gate P4 give the percentage of FRET-positive cells included in P2 according to the previous fluorescence-activated cell sorting (FACS) configuration for the different co-expression in living cells ( Figure 6 ); ( E ) Bar diagram summarizing the FRET-positive signals from four independent experiments ( n = 4). Statistical tests give a significance with a p

Techniques Used: Expressing, Fluorescence, FACS

BD FACSAria™ III cell sorter configuration and experimental setup of Förster resonance energy transfer (FRET)-measurements. Firstly, gate P1 selecting living cells (according to forward and sideward scatter FSC-A/SSC-A) and gate P2 selecting cells in P1 (according to forward and sideward scatter FSC-W/SSC-W in order to exclude joined or grouped cells), have been applied (not shown in this figure). Secondly, HEK293T cells expressing Clover or mRuby2 individually, in combination or as a fusion protein, were analyzed with three different filters in order to construct analysis gates and to define the FRET-positive signal. Clover (green fluorescent protein variant) was excited with the laser 488 nm and the FITC filter was used to examine fluorescence emission. mRuby2 (red fluorescent protein variant) was excited with the laser 561 nm and the PE-mCherry filter was used (panel A ). Importantly, mRuby2 and Clover showed some emission in the FRET-channel (PerCP-Cy 5.5 filter). Thus, a gate (P3) was constructed to exclude cells that emitted a false-positive signal in the FRET-channel (panel B ). As described in this image, cells co-transfected with Clover and mRuby2 exerted an aleatory FRET signal that should also be excluded. Therefore, an analysis gate (P4, in red) was applied to determine the FRET-positive cells when the FRET-adapted filters were selected (PerCP-Cy 5.5 and FITC filters, panel C ). This P4 gate excluded cells that were co-transfected with Clover and mRuby2 and thus are FRET-negative (0.5% of P2), while including cells that showed a FRET-positive signal (Clover fused mRuby2; 51.8% of P2). This gating strategy allowed for assessment the enhanced emission of the acceptor fluorochrome and therefore demonstrated the energy transfer between the two fluorochromes; namely the protein interactions.
Figure Legend Snippet: BD FACSAria™ III cell sorter configuration and experimental setup of Förster resonance energy transfer (FRET)-measurements. Firstly, gate P1 selecting living cells (according to forward and sideward scatter FSC-A/SSC-A) and gate P2 selecting cells in P1 (according to forward and sideward scatter FSC-W/SSC-W in order to exclude joined or grouped cells), have been applied (not shown in this figure). Secondly, HEK293T cells expressing Clover or mRuby2 individually, in combination or as a fusion protein, were analyzed with three different filters in order to construct analysis gates and to define the FRET-positive signal. Clover (green fluorescent protein variant) was excited with the laser 488 nm and the FITC filter was used to examine fluorescence emission. mRuby2 (red fluorescent protein variant) was excited with the laser 561 nm and the PE-mCherry filter was used (panel A ). Importantly, mRuby2 and Clover showed some emission in the FRET-channel (PerCP-Cy 5.5 filter). Thus, a gate (P3) was constructed to exclude cells that emitted a false-positive signal in the FRET-channel (panel B ). As described in this image, cells co-transfected with Clover and mRuby2 exerted an aleatory FRET signal that should also be excluded. Therefore, an analysis gate (P4, in red) was applied to determine the FRET-positive cells when the FRET-adapted filters were selected (PerCP-Cy 5.5 and FITC filters, panel C ). This P4 gate excluded cells that were co-transfected with Clover and mRuby2 and thus are FRET-negative (0.5% of P2), while including cells that showed a FRET-positive signal (Clover fused mRuby2; 51.8% of P2). This gating strategy allowed for assessment the enhanced emission of the acceptor fluorochrome and therefore demonstrated the energy transfer between the two fluorochromes; namely the protein interactions.

Techniques Used: Förster Resonance Energy Transfer, Expressing, Construct, Variant Assay, Fluorescence, Transfection

10) Product Images from "Molecular evolution of versatile derivatives from a GFP-like protein in the marine copepod Chiridius poppei"

Article Title: Molecular evolution of versatile derivatives from a GFP-like protein in the marine copepod Chiridius poppei

Journal: PLoS ONE

doi: 10.1371/journal.pone.0181186

FACS analysis of HCT116 cells expressing YGFP derivatives or commercial GFP proteins. Transfectants (TF) were selected using 1 μg/ml puromycin for 10 days, and fluorescence-positive cells were sorted by FACSaria III, as described in Materials and Methods. eYGFP opt and eYGFPuv opt nucleotide sequences are available from the DDBJ/EMBL/GenBank nucleotide sequence databases with accession numbers LC217534 (eYGFP opt ) and LC217535 (eYGFPuv opt ), respectively. Data are representative of three independent experiments.
Figure Legend Snippet: FACS analysis of HCT116 cells expressing YGFP derivatives or commercial GFP proteins. Transfectants (TF) were selected using 1 μg/ml puromycin for 10 days, and fluorescence-positive cells were sorted by FACSaria III, as described in Materials and Methods. eYGFP opt and eYGFPuv opt nucleotide sequences are available from the DDBJ/EMBL/GenBank nucleotide sequence databases with accession numbers LC217534 (eYGFP opt ) and LC217535 (eYGFPuv opt ), respectively. Data are representative of three independent experiments.

Techniques Used: FACS, Expressing, Fluorescence, Sequencing

11) Product Images from "PTEN-Regulated AID Transcription in Germinal Center B Cells Is Essential for the Class-Switch Recombination and IgG Antibody Responses"

Article Title: PTEN-Regulated AID Transcription in Germinal Center B Cells Is Essential for the Class-Switch Recombination and IgG Antibody Responses

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.00371

Repression of activation-induced cytidine deaminase (AID) induction in germinal center B cells (GCBs) from Pten fl/fl Cγ1 Cre/+ mice. (A) Flow cytometry sorting of in vitro induced IgM-BCR expressing splenic B cells from Pten +/+ Cγ1 Cre/+ and Pten fl/fl Cγ1 Cre/+ mice. Pure splenic B cells from control and KO mice (three mice for each group) were stimulated with LPS plus interleukin-4 (IL-4) for 4 days before sorting. (B) The expression levels of PTEN, p-AKT, p-FoxO1/3a, and AID were determined in Pten +/+ Cγ1 Cre/+ and Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells (left). Reduced PTEN, AID expression and enhanced p-AKT and p-FoxO1/3a expression were observed in Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells. The in vitro induced IgM-BCR expressing B cells were isolated by the FACSAria III Cell Sorter as described in (A) . Statistical comparison of protein expression level was also shown in right. Ratio values of different proteins from Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells were normalized to that of the Pten +/+ Cγ1 Cre/+ IgM-BCR expressing B cells. Data were given from one representative of at least three independent experiments. * p
Figure Legend Snippet: Repression of activation-induced cytidine deaminase (AID) induction in germinal center B cells (GCBs) from Pten fl/fl Cγ1 Cre/+ mice. (A) Flow cytometry sorting of in vitro induced IgM-BCR expressing splenic B cells from Pten +/+ Cγ1 Cre/+ and Pten fl/fl Cγ1 Cre/+ mice. Pure splenic B cells from control and KO mice (three mice for each group) were stimulated with LPS plus interleukin-4 (IL-4) for 4 days before sorting. (B) The expression levels of PTEN, p-AKT, p-FoxO1/3a, and AID were determined in Pten +/+ Cγ1 Cre/+ and Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells (left). Reduced PTEN, AID expression and enhanced p-AKT and p-FoxO1/3a expression were observed in Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells. The in vitro induced IgM-BCR expressing B cells were isolated by the FACSAria III Cell Sorter as described in (A) . Statistical comparison of protein expression level was also shown in right. Ratio values of different proteins from Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells were normalized to that of the Pten +/+ Cγ1 Cre/+ IgM-BCR expressing B cells. Data were given from one representative of at least three independent experiments. * p

Techniques Used: Activation Assay, Mouse Assay, Flow Cytometry, Cytometry, In Vitro, Expressing, Isolation

12) Product Images from "Utilization of leukapheresis and CD4 positive selection in Treg isolation and the ex-vivo expansion for a clinical application in transplantation and autoimmune disorders"

Article Title: Utilization of leukapheresis and CD4 positive selection in Treg isolation and the ex-vivo expansion for a clinical application in transplantation and autoimmune disorders

Journal: Oncotarget

doi: 10.18632/oncotarget.13101

Schema of gating strategy for Treg sorting Tregs were isolated in 2-step process: firstly, CD4 + cells were pre-enriched from leukapheresis product via immunomagnetic positive selection on CliniMACS ® device and then cells were stained with monoclonal antibodies and separated by Fluorescence Activated Cell Sorting (FACS). During FACS cells were gated as follows: ( A ) lymphocytes were identified on forward (FSC) and side-scatter (SSC) plot, ( B ) and ( C ) doublets were excluded by applying SSC-H vs SSC-W and FSC-H vs FSC-W gates, ( D ) followed by CD4 + cell gating. ( E ) Finally, Tregs and Teffectors were gated as CD25 hi CD127 lo/− and CD25 lo/− CD127 + cells, respectively; ( F ) dot-plot CD4 PerCP vs CD25 APC was used as a reference for correct Treg gating based on lower CD4 expression in Treg population than in Teffectors. ( G ) table with population hierarchy and statistics generated by FACSDiva Software during cell sorting on FACSAria III cell sorter (BD Biosciences, San Jose, CA, USA
Figure Legend Snippet: Schema of gating strategy for Treg sorting Tregs were isolated in 2-step process: firstly, CD4 + cells were pre-enriched from leukapheresis product via immunomagnetic positive selection on CliniMACS ® device and then cells were stained with monoclonal antibodies and separated by Fluorescence Activated Cell Sorting (FACS). During FACS cells were gated as follows: ( A ) lymphocytes were identified on forward (FSC) and side-scatter (SSC) plot, ( B ) and ( C ) doublets were excluded by applying SSC-H vs SSC-W and FSC-H vs FSC-W gates, ( D ) followed by CD4 + cell gating. ( E ) Finally, Tregs and Teffectors were gated as CD25 hi CD127 lo/− and CD25 lo/− CD127 + cells, respectively; ( F ) dot-plot CD4 PerCP vs CD25 APC was used as a reference for correct Treg gating based on lower CD4 expression in Treg population than in Teffectors. ( G ) table with population hierarchy and statistics generated by FACSDiva Software during cell sorting on FACSAria III cell sorter (BD Biosciences, San Jose, CA, USA

Techniques Used: Isolation, Selection, Staining, Fluorescence, FACS, Expressing, Generated, Software

13) Product Images from "Characterization of an attenuated SARS-CoV-2 variant with a deletion at the S1/S2 junction of the spike protein"

Article Title: Characterization of an attenuated SARS-CoV-2 variant with a deletion at the S1/S2 junction of the spike protein

Journal: Nature Communications

doi: 10.1038/s41467-021-23166-0

T cell response to Ca-DelMut immunization in Ad5-hACE2 transduced mice. A Timeline of Ad5-hACE2 transduction and immunization of Ad5-hACE2 transduced mice with Ca-DelMut. B Ca-DelMut induces IFN-γ positive CD4 + and CD8 + T cell responses in Ad5-hACE2 transduced mice. Four weeks after immunization, splenocytes and lung cells were obtained and stimulated with either spike or NP peptide pools or incubated without peptides overnight in the presence of BFA. Surface markers were stained, and cells fixed and permeabilized. Intracellular cytokines were then stained with antibodies. Sample data were acquired using a BD FACSAria III cell sorter. IFN-γ + CD4 + and CD8T cells in immunized ( n = 5) and naive groups ( n = 4) were compared. Statistical comparisons between means were performed by Student’s t -test (2-tailed): **** p
Figure Legend Snippet: T cell response to Ca-DelMut immunization in Ad5-hACE2 transduced mice. A Timeline of Ad5-hACE2 transduction and immunization of Ad5-hACE2 transduced mice with Ca-DelMut. B Ca-DelMut induces IFN-γ positive CD4 + and CD8 + T cell responses in Ad5-hACE2 transduced mice. Four weeks after immunization, splenocytes and lung cells were obtained and stimulated with either spike or NP peptide pools or incubated without peptides overnight in the presence of BFA. Surface markers were stained, and cells fixed and permeabilized. Intracellular cytokines were then stained with antibodies. Sample data were acquired using a BD FACSAria III cell sorter. IFN-γ + CD4 + and CD8T cells in immunized ( n = 5) and naive groups ( n = 4) were compared. Statistical comparisons between means were performed by Student’s t -test (2-tailed): **** p

Techniques Used: Mouse Assay, Transduction, Incubation, Staining

14) Product Images from "Single Amino Acid Substitution N659D in HIV-2 Envelope Glycoprotein (Env) Impairs Viral Release and Hampers BST-2 Antagonism"

Article Title: Single Amino Acid Substitution N659D in HIV-2 Envelope Glycoprotein (Env) Impairs Viral Release and Hampers BST-2 Antagonism

Journal: Viruses

doi: 10.3390/v8100285

HEK293T cells co-expressing the fusion proteins in the following combinations were analyzed with the configured BD FACSAria™ III cell sorter. ( A ) BST-2 fused Clover and HIV-2 Env WT fused to mRuby2; ( B ) BST-2 fused Clover and HIV-2 Env 1–749 fused mRuby2; ( C ) BST-2 fused Clover and HIV-2 Env N659D fused mRuby2 and ( D ) BST-2 fused Clover and HIV-2 Nef fused mRuby2. Numbers indicated in gate P4 give the percentage of FRET-positive cells included in P2 according to the previous fluorescence-activated cell sorting (FACS) configuration for the different co-expression in living cells ( Figure 6 ); ( E ) Bar diagram summarizing the FRET-positive signals from four independent experiments ( n = 4). Statistical tests give a significance with a p
Figure Legend Snippet: HEK293T cells co-expressing the fusion proteins in the following combinations were analyzed with the configured BD FACSAria™ III cell sorter. ( A ) BST-2 fused Clover and HIV-2 Env WT fused to mRuby2; ( B ) BST-2 fused Clover and HIV-2 Env 1–749 fused mRuby2; ( C ) BST-2 fused Clover and HIV-2 Env N659D fused mRuby2 and ( D ) BST-2 fused Clover and HIV-2 Nef fused mRuby2. Numbers indicated in gate P4 give the percentage of FRET-positive cells included in P2 according to the previous fluorescence-activated cell sorting (FACS) configuration for the different co-expression in living cells ( Figure 6 ); ( E ) Bar diagram summarizing the FRET-positive signals from four independent experiments ( n = 4). Statistical tests give a significance with a p

Techniques Used: Expressing, Fluorescence, FACS

BD FACSAria™ III cell sorter configuration and experimental setup of Förster resonance energy transfer (FRET)-measurements. Firstly, gate P1 selecting living cells (according to forward and sideward scatter FSC-A/SSC-A) and gate P2 selecting cells in P1 (according to forward and sideward scatter FSC-W/SSC-W in order to exclude joined or grouped cells), have been applied (not shown in this figure). Secondly, HEK293T cells expressing Clover or mRuby2 individually, in combination or as a fusion protein, were analyzed with three different filters in order to construct analysis gates and to define the FRET-positive signal. Clover (green fluorescent protein variant) was excited with the laser 488 nm and the FITC filter was used to examine fluorescence emission. mRuby2 (red fluorescent protein variant) was excited with the laser 561 nm and the PE-mCherry filter was used (panel A ). Importantly, mRuby2 and Clover showed some emission in the FRET-channel (PerCP-Cy 5.5 filter). Thus, a gate (P3) was constructed to exclude cells that emitted a false-positive signal in the FRET-channel (panel B ). As described in this image, cells co-transfected with Clover and mRuby2 exerted an aleatory FRET signal that should also be excluded. Therefore, an analysis gate (P4, in red) was applied to determine the FRET-positive cells when the FRET-adapted filters were selected (PerCP-Cy 5.5 and FITC filters, panel C ). This P4 gate excluded cells that were co-transfected with Clover and mRuby2 and thus are FRET-negative (0.5% of P2), while including cells that showed a FRET-positive signal (Clover fused mRuby2; 51.8% of P2). This gating strategy allowed for assessment the enhanced emission of the acceptor fluorochrome and therefore demonstrated the energy transfer between the two fluorochromes; namely the protein interactions.
Figure Legend Snippet: BD FACSAria™ III cell sorter configuration and experimental setup of Förster resonance energy transfer (FRET)-measurements. Firstly, gate P1 selecting living cells (according to forward and sideward scatter FSC-A/SSC-A) and gate P2 selecting cells in P1 (according to forward and sideward scatter FSC-W/SSC-W in order to exclude joined or grouped cells), have been applied (not shown in this figure). Secondly, HEK293T cells expressing Clover or mRuby2 individually, in combination or as a fusion protein, were analyzed with three different filters in order to construct analysis gates and to define the FRET-positive signal. Clover (green fluorescent protein variant) was excited with the laser 488 nm and the FITC filter was used to examine fluorescence emission. mRuby2 (red fluorescent protein variant) was excited with the laser 561 nm and the PE-mCherry filter was used (panel A ). Importantly, mRuby2 and Clover showed some emission in the FRET-channel (PerCP-Cy 5.5 filter). Thus, a gate (P3) was constructed to exclude cells that emitted a false-positive signal in the FRET-channel (panel B ). As described in this image, cells co-transfected with Clover and mRuby2 exerted an aleatory FRET signal that should also be excluded. Therefore, an analysis gate (P4, in red) was applied to determine the FRET-positive cells when the FRET-adapted filters were selected (PerCP-Cy 5.5 and FITC filters, panel C ). This P4 gate excluded cells that were co-transfected with Clover and mRuby2 and thus are FRET-negative (0.5% of P2), while including cells that showed a FRET-positive signal (Clover fused mRuby2; 51.8% of P2). This gating strategy allowed for assessment the enhanced emission of the acceptor fluorochrome and therefore demonstrated the energy transfer between the two fluorochromes; namely the protein interactions.

Techniques Used: Förster Resonance Energy Transfer, Expressing, Construct, Variant Assay, Fluorescence, Transfection

15) Product Images from "Investigating the potential of Shikonin as a novel hypertrophic scar treatment"

Article Title: Investigating the potential of Shikonin as a novel hypertrophic scar treatment

Journal: Journal of Biomedical Science

doi: 10.1186/s12929-015-0172-9

Apoptosis in Kc and HSF determined by flow cytometry. a Apoptosis rate in Kc and HSF following SHI treatment; b Quantitative analysis of SHI-induced apoptosis in Kc and HSF. Kc and HSF were treated with 0, 1 or 3 μg/mL SHI for 12 h and then stained with annexin V and propidium iodide as per the manufacturer’s instructions. Flow cytometry was performed using FACSAria™ III Cell Sorter (Becton Dickinson). All experiments were performed three times using cells from 3 patients. Triplicate treatments were assessed in cells from each patient. Quantitative data from the 3 patients were pooled. * p
Figure Legend Snippet: Apoptosis in Kc and HSF determined by flow cytometry. a Apoptosis rate in Kc and HSF following SHI treatment; b Quantitative analysis of SHI-induced apoptosis in Kc and HSF. Kc and HSF were treated with 0, 1 or 3 μg/mL SHI for 12 h and then stained with annexin V and propidium iodide as per the manufacturer’s instructions. Flow cytometry was performed using FACSAria™ III Cell Sorter (Becton Dickinson). All experiments were performed three times using cells from 3 patients. Triplicate treatments were assessed in cells from each patient. Quantitative data from the 3 patients were pooled. * p

Techniques Used: Flow Cytometry, Cytometry, Staining

Related Articles

Staining:

Article Title: Interferons and viruses induce a novel truncated ACE2 isoform and not the full-length SARS-CoV-2 receptor.
Article Snippet: T24 cells were transiently transfected with ACE2–GFP or dACE2–Myc, or co-transfected with both constructs in 12-well plates (1 × 104 cells per well). .. After 24 h, cells were stained with recombinant biotinylated spike protein RBD as described above and analyzed with multiparametric flow cytometry on a FACS Aria III (BD Biosciences) and FlowJo v10 software (BD Biosciences). .. Carboxypeptidase activity assays.

Article Title: ACME dissociation: a versatile cell fixation-dissociation method for single-cell transcriptomics
Article Snippet: Staining concentrations require optimization and ultimately depend on FACS/Cytometer adjustments and cell concentration. .. Cells were stained in the dark, on ice, for 30–45 min, and visualized using a CytoFlex S Flow Cytometer (Beckman Coulter) or sorted using a BD FACS Aria III (BD Biosciences) Cell Sorter. .. To avoid RNAse contamination during cell sorting, the FACS was thoroughly decontaminated with bleach and pre-cooled before sorting, keeping injection and collection chambers at 4 °C during the process.

Recombinant:

Article Title: Interferons and viruses induce a novel truncated ACE2 isoform and not the full-length SARS-CoV-2 receptor.
Article Snippet: T24 cells were transiently transfected with ACE2–GFP or dACE2–Myc, or co-transfected with both constructs in 12-well plates (1 × 104 cells per well). .. After 24 h, cells were stained with recombinant biotinylated spike protein RBD as described above and analyzed with multiparametric flow cytometry on a FACS Aria III (BD Biosciences) and FlowJo v10 software (BD Biosciences). .. Carboxypeptidase activity assays.

Flow Cytometry:

Article Title: Interferons and viruses induce a novel truncated ACE2 isoform and not the full-length SARS-CoV-2 receptor.
Article Snippet: T24 cells were transiently transfected with ACE2–GFP or dACE2–Myc, or co-transfected with both constructs in 12-well plates (1 × 104 cells per well). .. After 24 h, cells were stained with recombinant biotinylated spike protein RBD as described above and analyzed with multiparametric flow cytometry on a FACS Aria III (BD Biosciences) and FlowJo v10 software (BD Biosciences). .. Carboxypeptidase activity assays.

Article Title: ACME dissociation: a versatile cell fixation-dissociation method for single-cell transcriptomics
Article Snippet: Staining concentrations require optimization and ultimately depend on FACS/Cytometer adjustments and cell concentration. .. Cells were stained in the dark, on ice, for 30–45 min, and visualized using a CytoFlex S Flow Cytometer (Beckman Coulter) or sorted using a BD FACS Aria III (BD Biosciences) Cell Sorter. .. To avoid RNAse contamination during cell sorting, the FACS was thoroughly decontaminated with bleach and pre-cooled before sorting, keeping injection and collection chambers at 4 °C during the process.

Article Title: CD200–CD200R immune checkpoint engagement regulates ILC2 effector function and ameliorates lung inflammation in asthma
Article Snippet: Data were analyzed with FlowJo software (TreeStar) version 10. .. ILC2 sorting and ex vivo stimulation All cells were purified by flow cytometry using BD FACS ARIA III (BD biosciences, San Jose, CA) with a purity of > 95%. .. The isolated ILC2s were cultured (at least 5 × 103 cells/well) in 96-well round-bottom plates with Gibco™ Roswell Park Memorial Institute (RPMI) 1640 medium (Thermo Fisher Scientific, Waltham, MA) that was supplemented with 10% fetal bovine serum (FBS), 2% antibiotics (penicillin and streptomycin), and 0.05 mM b-mercaptoethanol.

FACS:

Article Title: Interferons and viruses induce a novel truncated ACE2 isoform and not the full-length SARS-CoV-2 receptor.
Article Snippet: T24 cells were transiently transfected with ACE2–GFP or dACE2–Myc, or co-transfected with both constructs in 12-well plates (1 × 104 cells per well). .. After 24 h, cells were stained with recombinant biotinylated spike protein RBD as described above and analyzed with multiparametric flow cytometry on a FACS Aria III (BD Biosciences) and FlowJo v10 software (BD Biosciences). .. Carboxypeptidase activity assays.

Article Title: ACME dissociation: a versatile cell fixation-dissociation method for single-cell transcriptomics
Article Snippet: Staining concentrations require optimization and ultimately depend on FACS/Cytometer adjustments and cell concentration. .. Cells were stained in the dark, on ice, for 30–45 min, and visualized using a CytoFlex S Flow Cytometer (Beckman Coulter) or sorted using a BD FACS Aria III (BD Biosciences) Cell Sorter. .. To avoid RNAse contamination during cell sorting, the FACS was thoroughly decontaminated with bleach and pre-cooled before sorting, keeping injection and collection chambers at 4 °C during the process.

Article Title: CD200–CD200R immune checkpoint engagement regulates ILC2 effector function and ameliorates lung inflammation in asthma
Article Snippet: Data were analyzed with FlowJo software (TreeStar) version 10. .. ILC2 sorting and ex vivo stimulation All cells were purified by flow cytometry using BD FACS ARIA III (BD biosciences, San Jose, CA) with a purity of > 95%. .. The isolated ILC2s were cultured (at least 5 × 103 cells/well) in 96-well round-bottom plates with Gibco™ Roswell Park Memorial Institute (RPMI) 1640 medium (Thermo Fisher Scientific, Waltham, MA) that was supplemented with 10% fetal bovine serum (FBS), 2% antibiotics (penicillin and streptomycin), and 0.05 mM b-mercaptoethanol.

Article Title: GATA transcription factors, SOX17 and TFAP2C, drive the human germ-cell specification program
Article Snippet: The images of the aggregates were taken under an M205C stereo microscope (Leica Microsystems) equipped with a DP72 CCD camera and DP2-BSW software (Olympus). .. FACSFor analysis of the cellular contents of the aggregates, the aggregates were collected on the designated days of induction, washed once in PBS, and dissociated with 0.25% Trypsin–EDTA for 10–15 min at 37°C with gentle pipetting every 5 min. Trypsin was neutralized with a 5× volume of 10% FBS in DMEM, and the resuspended cells were processed with FACS Aria III system (BD Biosciences) and analyzed with FACS Diva software. .. The method for selecting CRISPR-mediated knockout clones is described in the section “Generation of knockout lines.”.

Article Title: Zeb1-Hdac2-eNOS circuitry identifies early cardiovascular precursors in naive mouse embryonic stem cells
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Article Title: The Function of FK506-Binding Protein 13 in Protein Quality Control Protects Plasma Cells from Endoplasmic Reticulum Stress-Associated Apoptosis
Article Snippet: .. Enzyme-Linked Immunosorbent Assays (ELISA)Sixteen hours after transfection of J558 cells with MigR1, MigR1-myc-FKBP13, pGFP-V-RS, or pGFP-V-RS-shFKBP13, GFP-positive cells were sorted using a FACS Aria III (BD Biosciences) and cultured in DMEM with 10% horse serum. .. After 48 h, supernatants were collected and assayed by sandwich ELISA to measure secreted IgA.

Article Title: The transcription factor EGR2 is the molecular linchpin connecting STAT6 activation to the late, stable epigenomic program of alternative macrophage polarization
Article Snippet: For intracellular detection of RELMα, Zenon Alexa fluor 350 rabbit IgG labeling kit (Thermo Fischer Scientific) was used to add fluorescent label to RELMα (Peprotech) antibody, BD Cytofix Cytoperm fixation/permeabilization solution kit (BD Biosciences) was used for fixation/permeabilization and staining according to the manufacturer's recommendations. .. Samples were acquired on a BD FACS Aria III (BD Biosciences) cell sorter using BD FACSDiva 6.0 software (BD Biosciences). .. Data analysis was performed using FlowJo v10 software (Becton Dickinson and Company).

Software:

Article Title: Interferons and viruses induce a novel truncated ACE2 isoform and not the full-length SARS-CoV-2 receptor.
Article Snippet: T24 cells were transiently transfected with ACE2–GFP or dACE2–Myc, or co-transfected with both constructs in 12-well plates (1 × 104 cells per well). .. After 24 h, cells were stained with recombinant biotinylated spike protein RBD as described above and analyzed with multiparametric flow cytometry on a FACS Aria III (BD Biosciences) and FlowJo v10 software (BD Biosciences). .. Carboxypeptidase activity assays.

Article Title: GATA transcription factors, SOX17 and TFAP2C, drive the human germ-cell specification program
Article Snippet: The images of the aggregates were taken under an M205C stereo microscope (Leica Microsystems) equipped with a DP72 CCD camera and DP2-BSW software (Olympus). .. FACSFor analysis of the cellular contents of the aggregates, the aggregates were collected on the designated days of induction, washed once in PBS, and dissociated with 0.25% Trypsin–EDTA for 10–15 min at 37°C with gentle pipetting every 5 min. Trypsin was neutralized with a 5× volume of 10% FBS in DMEM, and the resuspended cells were processed with FACS Aria III system (BD Biosciences) and analyzed with FACS Diva software. .. The method for selecting CRISPR-mediated knockout clones is described in the section “Generation of knockout lines.”.

Article Title: The transcription factor EGR2 is the molecular linchpin connecting STAT6 activation to the late, stable epigenomic program of alternative macrophage polarization
Article Snippet: For intracellular detection of RELMα, Zenon Alexa fluor 350 rabbit IgG labeling kit (Thermo Fischer Scientific) was used to add fluorescent label to RELMα (Peprotech) antibody, BD Cytofix Cytoperm fixation/permeabilization solution kit (BD Biosciences) was used for fixation/permeabilization and staining according to the manufacturer's recommendations. .. Samples were acquired on a BD FACS Aria III (BD Biosciences) cell sorter using BD FACSDiva 6.0 software (BD Biosciences). .. Data analysis was performed using FlowJo v10 software (Becton Dickinson and Company).

Ex Vivo:

Article Title: CD200–CD200R immune checkpoint engagement regulates ILC2 effector function and ameliorates lung inflammation in asthma
Article Snippet: Data were analyzed with FlowJo software (TreeStar) version 10. .. ILC2 sorting and ex vivo stimulation All cells were purified by flow cytometry using BD FACS ARIA III (BD biosciences, San Jose, CA) with a purity of > 95%. .. The isolated ILC2s were cultured (at least 5 × 103 cells/well) in 96-well round-bottom plates with Gibco™ Roswell Park Memorial Institute (RPMI) 1640 medium (Thermo Fisher Scientific, Waltham, MA) that was supplemented with 10% fetal bovine serum (FBS), 2% antibiotics (penicillin and streptomycin), and 0.05 mM b-mercaptoethanol.

Purification:

Article Title: CD200–CD200R immune checkpoint engagement regulates ILC2 effector function and ameliorates lung inflammation in asthma
Article Snippet: Data were analyzed with FlowJo software (TreeStar) version 10. .. ILC2 sorting and ex vivo stimulation All cells were purified by flow cytometry using BD FACS ARIA III (BD biosciences, San Jose, CA) with a purity of > 95%. .. The isolated ILC2s were cultured (at least 5 × 103 cells/well) in 96-well round-bottom plates with Gibco™ Roswell Park Memorial Institute (RPMI) 1640 medium (Thermo Fisher Scientific, Waltham, MA) that was supplemented with 10% fetal bovine serum (FBS), 2% antibiotics (penicillin and streptomycin), and 0.05 mM b-mercaptoethanol.

Enzyme-linked Immunosorbent Assay:

Article Title: The Function of FK506-Binding Protein 13 in Protein Quality Control Protects Plasma Cells from Endoplasmic Reticulum Stress-Associated Apoptosis
Article Snippet: .. Enzyme-Linked Immunosorbent Assays (ELISA)Sixteen hours after transfection of J558 cells with MigR1, MigR1-myc-FKBP13, pGFP-V-RS, or pGFP-V-RS-shFKBP13, GFP-positive cells were sorted using a FACS Aria III (BD Biosciences) and cultured in DMEM with 10% horse serum. .. After 48 h, supernatants were collected and assayed by sandwich ELISA to measure secreted IgA.

Transfection:

Article Title: The Function of FK506-Binding Protein 13 in Protein Quality Control Protects Plasma Cells from Endoplasmic Reticulum Stress-Associated Apoptosis
Article Snippet: .. Enzyme-Linked Immunosorbent Assays (ELISA)Sixteen hours after transfection of J558 cells with MigR1, MigR1-myc-FKBP13, pGFP-V-RS, or pGFP-V-RS-shFKBP13, GFP-positive cells were sorted using a FACS Aria III (BD Biosciences) and cultured in DMEM with 10% horse serum. .. After 48 h, supernatants were collected and assayed by sandwich ELISA to measure secreted IgA.

Cell Culture:

Article Title: The Function of FK506-Binding Protein 13 in Protein Quality Control Protects Plasma Cells from Endoplasmic Reticulum Stress-Associated Apoptosis
Article Snippet: .. Enzyme-Linked Immunosorbent Assays (ELISA)Sixteen hours after transfection of J558 cells with MigR1, MigR1-myc-FKBP13, pGFP-V-RS, or pGFP-V-RS-shFKBP13, GFP-positive cells were sorted using a FACS Aria III (BD Biosciences) and cultured in DMEM with 10% horse serum. .. After 48 h, supernatants were collected and assayed by sandwich ELISA to measure secreted IgA.

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    Becton Dickinson facs system
    Generation and characterization of EpCAM-specific CAR-NK-92 cells. (a) Structure diagram of EpCAM-specific CAR. EF1 α , promoter; SP, signal peptide; scFv, single-chain variable fragment. (b) Transduction efficiency of lentivirus in NK-92 cells. NK-92 cells were transduced with empty lentivirus vector (Ctrl-NK-92) or lentivirus containing the EpCAM-specific CAR encoding sequence (CAR-NK-92) and sorted by a <t>FACS</t> machine with a <t>GFP</t> marker. The GFP-positive rate of transduced NK-92 cells after sorting is shown. (c) Western blot analysis of the CAR expression in NK-92 cells with a monoclonal anti-human CD3 ζ antibody. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was also detected as an internal control.
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    Repression of activation-induced cytidine deaminase (AID) induction in germinal center B cells (GCBs) from Pten fl/fl Cγ1 Cre/+ mice. (A) Flow cytometry sorting of in vitro induced IgM-BCR expressing splenic B cells from Pten +/+ Cγ1 Cre/+ and Pten fl/fl Cγ1 Cre/+ mice. Pure splenic B cells from control and KO mice (three mice for each group) were stimulated with LPS plus interleukin-4 (IL-4) for 4 days before sorting. (B) The expression levels of PTEN, p-AKT, p-FoxO1/3a, and AID were determined in Pten +/+ Cγ1 Cre/+ and Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells (left). Reduced PTEN, AID expression and enhanced p-AKT and p-FoxO1/3a expression were observed in Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells. The in vitro induced IgM-BCR expressing B cells were isolated by the <t>FACSAria</t> <t>III</t> Cell Sorter as described in (A) . Statistical comparison of protein expression level was also shown in right. Ratio values of different proteins from Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells were normalized to that of the Pten +/+ Cγ1 Cre/+ IgM-BCR expressing B cells. Data were given from one representative of at least three independent experiments. * p
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    NPC treatment alters the gene expression signature of CNS-infiltrating inflammatory MCs in EAE. ( A ) Cohorts of 4–7 MOG 35–55 -immunized C57BL/6 mice intrathecally treated with either PBS or NPCs at the peak of the disease (2–4 days after clinical onset). At 7 days after transplantation, CNS tissues were pooled and CNS-infiltrating MCs were <t>FACS-sorted</t> according to the phenotype CD45 hi Ly6G – <t>CD11b</t> + Ly6C hi MHC-II + . Sorting strategy used for 3 independent FACS sorting experiments is shown. ( B ) Next-generation sequencing was performed on RNA extracted from sorted cells of 3 independent experiments and respective CNS harvests. Six hundred ten genes that are significantly altered in 3 different statistical tests to a minimum significance threshold of P ≤ 0.01 are shown in the heatmap. ( C ) Volcano plot showing the fold change and significance of genes in MCs from PBS- versus NPC-treated EAE mice. ( D ). Black and gray nodes represent enriched pathways with sizes corresponding to FDR-adjusted enrichment P value ( P ≤ 0.05). Red dots represent upregulated genes and blue dots downregulated genes, whereas the dot size indicates significance ( P ≤ 0.01).
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    RNA-seq of stained and sorted cell populations A : Bioanalyzer profiles of total RNA derived from unprocessed cells (harvested directly from trypsinised cells) compared to cells that have undergone glyoxal fixation, permeabilisation with 100% methanol and indirect immunofluorescence for CCNB1. 0.1-5 ng of total RNA was separated on total RNA pico chips on Agilent 2100 Bioanalyzer. B : Scatter plots comparing the normalised read count for each annotated gene in GRCh38 in poly(A)+ RNA-seq libraries derived from unprocessed and from glyoxal fixed, permeabilised and stained cells. Two biological replicates were sequenced for each condition and averaged, read counts were normalised to the total number of reads in each library. Data is shown for both COLO205 and MCF-7 cells. C : Flow cytometry density plots for MCF-7 cells labelled with anti-CCNB1 primary antibody and donkey Alexa Fluor-488 conjugated secondary antibody and sorted using a BD <t>FACSAria</t> <t>III</t> sorter. Intact cells (shown within the elliptical gate) were distinguished from off-scale events and cell debris using forward scatter (FSC) and side scatter (SSC) measurements (panel 1). Doublets were excluded from the gated cells by a 2-step gating strategy with pulse height (H) plotted against area (A) for FSC parameter followed by SSC-H versus SSC-A plot (panel 3). Fluorescence thresholds for isolation of CCNB1 positive and negative cell fractions are shown in panel 4. Gates were set using the negative control and the CCNB1 positive and negative sorting gates were moved apart from each other to maximise purity when sorted. D : Fluorescence histogram of DAPI intensities for sorted CCNB1 negative and positive populations E : Scatter plots comparing the normalised read count for each annotated gene in GRCh38 in poly(A)+ RNA-seq libraries derived from two biological replicates each of CCNB1 positive (left) and negative (right) MCF-7 cells. Note the particularly tight correlation after sorting towards the antigen of interest. E : Hierarchical clustering analysis of the 161 significantly different expressed genes identified DEseq2 analysis (p
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    Generation and characterization of EpCAM-specific CAR-NK-92 cells. (a) Structure diagram of EpCAM-specific CAR. EF1 α , promoter; SP, signal peptide; scFv, single-chain variable fragment. (b) Transduction efficiency of lentivirus in NK-92 cells. NK-92 cells were transduced with empty lentivirus vector (Ctrl-NK-92) or lentivirus containing the EpCAM-specific CAR encoding sequence (CAR-NK-92) and sorted by a FACS machine with a GFP marker. The GFP-positive rate of transduced NK-92 cells after sorting is shown. (c) Western blot analysis of the CAR expression in NK-92 cells with a monoclonal anti-human CD3 ζ antibody. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was also detected as an internal control.

    Journal: Journal of Immunology Research

    Article Title: Combination Therapy with EpCAM-CAR-NK-92 Cells and Regorafenib against Human Colorectal Cancer Models

    doi: 10.1155/2018/4263520

    Figure Lengend Snippet: Generation and characterization of EpCAM-specific CAR-NK-92 cells. (a) Structure diagram of EpCAM-specific CAR. EF1 α , promoter; SP, signal peptide; scFv, single-chain variable fragment. (b) Transduction efficiency of lentivirus in NK-92 cells. NK-92 cells were transduced with empty lentivirus vector (Ctrl-NK-92) or lentivirus containing the EpCAM-specific CAR encoding sequence (CAR-NK-92) and sorted by a FACS machine with a GFP marker. The GFP-positive rate of transduced NK-92 cells after sorting is shown. (c) Western blot analysis of the CAR expression in NK-92 cells with a monoclonal anti-human CD3 ζ antibody. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was also detected as an internal control.

    Article Snippet: After expansion for several days, GFP+ NK-92 cells were sorted with a FACS system (FACSAria III, Becton-Dickinson, USA).

    Techniques: Transduction, Plasmid Preparation, Sequencing, FACS, Marker, Western Blot, Expressing

    Repression of activation-induced cytidine deaminase (AID) induction in germinal center B cells (GCBs) from Pten fl/fl Cγ1 Cre/+ mice. (A) Flow cytometry sorting of in vitro induced IgM-BCR expressing splenic B cells from Pten +/+ Cγ1 Cre/+ and Pten fl/fl Cγ1 Cre/+ mice. Pure splenic B cells from control and KO mice (three mice for each group) were stimulated with LPS plus interleukin-4 (IL-4) for 4 days before sorting. (B) The expression levels of PTEN, p-AKT, p-FoxO1/3a, and AID were determined in Pten +/+ Cγ1 Cre/+ and Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells (left). Reduced PTEN, AID expression and enhanced p-AKT and p-FoxO1/3a expression were observed in Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells. The in vitro induced IgM-BCR expressing B cells were isolated by the FACSAria III Cell Sorter as described in (A) . Statistical comparison of protein expression level was also shown in right. Ratio values of different proteins from Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells were normalized to that of the Pten +/+ Cγ1 Cre/+ IgM-BCR expressing B cells. Data were given from one representative of at least three independent experiments. * p

    Journal: Frontiers in Immunology

    Article Title: PTEN-Regulated AID Transcription in Germinal Center B Cells Is Essential for the Class-Switch Recombination and IgG Antibody Responses

    doi: 10.3389/fimmu.2018.00371

    Figure Lengend Snippet: Repression of activation-induced cytidine deaminase (AID) induction in germinal center B cells (GCBs) from Pten fl/fl Cγ1 Cre/+ mice. (A) Flow cytometry sorting of in vitro induced IgM-BCR expressing splenic B cells from Pten +/+ Cγ1 Cre/+ and Pten fl/fl Cγ1 Cre/+ mice. Pure splenic B cells from control and KO mice (three mice for each group) were stimulated with LPS plus interleukin-4 (IL-4) for 4 days before sorting. (B) The expression levels of PTEN, p-AKT, p-FoxO1/3a, and AID were determined in Pten +/+ Cγ1 Cre/+ and Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells (left). Reduced PTEN, AID expression and enhanced p-AKT and p-FoxO1/3a expression were observed in Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells. The in vitro induced IgM-BCR expressing B cells were isolated by the FACSAria III Cell Sorter as described in (A) . Statistical comparison of protein expression level was also shown in right. Ratio values of different proteins from Pten fl/fl Cγ1 Cre/+ IgM-BCR expressing B cells were normalized to that of the Pten +/+ Cγ1 Cre/+ IgM-BCR expressing B cells. Data were given from one representative of at least three independent experiments. * p

    Article Snippet: Pre-incubated IgM-BCR expressing B cells were sorted by using FACSAria III Cell Sorter (BD).

    Techniques: Activation Assay, Mouse Assay, Flow Cytometry, Cytometry, In Vitro, Expressing, Isolation

    NPC treatment alters the gene expression signature of CNS-infiltrating inflammatory MCs in EAE. ( A ) Cohorts of 4–7 MOG 35–55 -immunized C57BL/6 mice intrathecally treated with either PBS or NPCs at the peak of the disease (2–4 days after clinical onset). At 7 days after transplantation, CNS tissues were pooled and CNS-infiltrating MCs were FACS-sorted according to the phenotype CD45 hi Ly6G – CD11b + Ly6C hi MHC-II + . Sorting strategy used for 3 independent FACS sorting experiments is shown. ( B ) Next-generation sequencing was performed on RNA extracted from sorted cells of 3 independent experiments and respective CNS harvests. Six hundred ten genes that are significantly altered in 3 different statistical tests to a minimum significance threshold of P ≤ 0.01 are shown in the heatmap. ( C ) Volcano plot showing the fold change and significance of genes in MCs from PBS- versus NPC-treated EAE mice. ( D ). Black and gray nodes represent enriched pathways with sizes corresponding to FDR-adjusted enrichment P value ( P ≤ 0.05). Red dots represent upregulated genes and blue dots downregulated genes, whereas the dot size indicates significance ( P ≤ 0.01).

    Journal: The Journal of Clinical Investigation

    Article Title: Neural precursor cell–secreted TGF- β2 redirects inflammatory monocyte-derived cells in CNS autoimmunity

    doi: 10.1172/JCI92387

    Figure Lengend Snippet: NPC treatment alters the gene expression signature of CNS-infiltrating inflammatory MCs in EAE. ( A ) Cohorts of 4–7 MOG 35–55 -immunized C57BL/6 mice intrathecally treated with either PBS or NPCs at the peak of the disease (2–4 days after clinical onset). At 7 days after transplantation, CNS tissues were pooled and CNS-infiltrating MCs were FACS-sorted according to the phenotype CD45 hi Ly6G – CD11b + Ly6C hi MHC-II + . Sorting strategy used for 3 independent FACS sorting experiments is shown. ( B ) Next-generation sequencing was performed on RNA extracted from sorted cells of 3 independent experiments and respective CNS harvests. Six hundred ten genes that are significantly altered in 3 different statistical tests to a minimum significance threshold of P ≤ 0.01 are shown in the heatmap. ( C ) Volcano plot showing the fold change and significance of genes in MCs from PBS- versus NPC-treated EAE mice. ( D ). Black and gray nodes represent enriched pathways with sizes corresponding to FDR-adjusted enrichment P value ( P ≤ 0.05). Red dots represent upregulated genes and blue dots downregulated genes, whereas the dot size indicates significance ( P ≤ 0.01).

    Article Snippet: Seven days after transplantation, CD45hi CD11b+ Ly6Chi Ly6G– MHC-II+ MCs were FACS-sorted (BD FACSAria III) from hindbrain and spinal cord as described above.

    Techniques: Expressing, Mouse Assay, Transplantation Assay, FACS, Next-Generation Sequencing

    RNA-seq of stained and sorted cell populations A : Bioanalyzer profiles of total RNA derived from unprocessed cells (harvested directly from trypsinised cells) compared to cells that have undergone glyoxal fixation, permeabilisation with 100% methanol and indirect immunofluorescence for CCNB1. 0.1-5 ng of total RNA was separated on total RNA pico chips on Agilent 2100 Bioanalyzer. B : Scatter plots comparing the normalised read count for each annotated gene in GRCh38 in poly(A)+ RNA-seq libraries derived from unprocessed and from glyoxal fixed, permeabilised and stained cells. Two biological replicates were sequenced for each condition and averaged, read counts were normalised to the total number of reads in each library. Data is shown for both COLO205 and MCF-7 cells. C : Flow cytometry density plots for MCF-7 cells labelled with anti-CCNB1 primary antibody and donkey Alexa Fluor-488 conjugated secondary antibody and sorted using a BD FACSAria III sorter. Intact cells (shown within the elliptical gate) were distinguished from off-scale events and cell debris using forward scatter (FSC) and side scatter (SSC) measurements (panel 1). Doublets were excluded from the gated cells by a 2-step gating strategy with pulse height (H) plotted against area (A) for FSC parameter followed by SSC-H versus SSC-A plot (panel 3). Fluorescence thresholds for isolation of CCNB1 positive and negative cell fractions are shown in panel 4. Gates were set using the negative control and the CCNB1 positive and negative sorting gates were moved apart from each other to maximise purity when sorted. D : Fluorescence histogram of DAPI intensities for sorted CCNB1 negative and positive populations E : Scatter plots comparing the normalised read count for each annotated gene in GRCh38 in poly(A)+ RNA-seq libraries derived from two biological replicates each of CCNB1 positive (left) and negative (right) MCF-7 cells. Note the particularly tight correlation after sorting towards the antigen of interest. E : Hierarchical clustering analysis of the 161 significantly different expressed genes identified DEseq2 analysis (p

    Journal: bioRxiv

    Article Title: Glyoxal fixation facilitates transcriptome analysis after antigen staining and cell sorting by flow cytometry

    doi: 10.1101/2020.10.05.326082

    Figure Lengend Snippet: RNA-seq of stained and sorted cell populations A : Bioanalyzer profiles of total RNA derived from unprocessed cells (harvested directly from trypsinised cells) compared to cells that have undergone glyoxal fixation, permeabilisation with 100% methanol and indirect immunofluorescence for CCNB1. 0.1-5 ng of total RNA was separated on total RNA pico chips on Agilent 2100 Bioanalyzer. B : Scatter plots comparing the normalised read count for each annotated gene in GRCh38 in poly(A)+ RNA-seq libraries derived from unprocessed and from glyoxal fixed, permeabilised and stained cells. Two biological replicates were sequenced for each condition and averaged, read counts were normalised to the total number of reads in each library. Data is shown for both COLO205 and MCF-7 cells. C : Flow cytometry density plots for MCF-7 cells labelled with anti-CCNB1 primary antibody and donkey Alexa Fluor-488 conjugated secondary antibody and sorted using a BD FACSAria III sorter. Intact cells (shown within the elliptical gate) were distinguished from off-scale events and cell debris using forward scatter (FSC) and side scatter (SSC) measurements (panel 1). Doublets were excluded from the gated cells by a 2-step gating strategy with pulse height (H) plotted against area (A) for FSC parameter followed by SSC-H versus SSC-A plot (panel 3). Fluorescence thresholds for isolation of CCNB1 positive and negative cell fractions are shown in panel 4. Gates were set using the negative control and the CCNB1 positive and negative sorting gates were moved apart from each other to maximise purity when sorted. D : Fluorescence histogram of DAPI intensities for sorted CCNB1 negative and positive populations E : Scatter plots comparing the normalised read count for each annotated gene in GRCh38 in poly(A)+ RNA-seq libraries derived from two biological replicates each of CCNB1 positive (left) and negative (right) MCF-7 cells. Note the particularly tight correlation after sorting towards the antigen of interest. E : Hierarchical clustering analysis of the 161 significantly different expressed genes identified DEseq2 analysis (p

    Article Snippet: Cells were sorted using a 100 μm nozzle (at 20PSI) on a BD FACSARIA III cell sorter (BD Biosciences, UK) with cooling of the sample and collection chambers enabled.

    Techniques: RNA Sequencing Assay, Staining, Derivative Assay, Immunofluorescence, Flow Cytometry, Fluorescence, Isolation, Negative Control