fbs  (Biochrom)

Journal: Life Science Alliance

Article Title: Immunoglobulin expression in the endoplasmic reticulum shapes the metabolic fitness of B lymphocytes

doi: 10.26508/lsa.202000700

Figure Lengend Snippet: BCR ablation leads to reduced metabolic flexibility. (A) Pictures show Mito Tracker Red-CMXRos staining alone (upper panels) and overlaid with transmitted light (lower panels) of WT and KO cells (magnification 63×, scale bar = 5 μm). (B) Cells were plated on d0 with or without oligomycin and cell numbers were assessed on d1 and d2 using the CCK8-kit. Values were normalized to the measurement obtained on d1. Statistical significance was determines using the ANOVA test. N = 7; * P = 0.0213 and 0.0341, ** P = 0.0012. (C) Cells were left untreated or incubated with oligomycin overnight. Cells were resupsended in medium containing pyruvate, glutamine, and glucose, and OCR was measured using Seahorse flux technology. One of three independent experiments is shown. a, oligomycin, b, FCCP, c, rotenone + antimycin. (D) Cells were cultured in glucose free RPMI+ dialyzed FBS in the presence of 10 mM glucose, 10 mM galactose, or 10 mM GlutaMAX with or without oligomycin. Cell numbers were assessed using the CCK8 kit after overnight incubation. Results are normalized to the values obtained from the untreated sample cultured with glucose. Differences between the indicated pairs of samples were tested for significance using the unpaired t test. N = 3; * P = 0.0242, ** P = 0.0033 and 0.0059, *** P = 0.0005 and 0.0002. KO, BCR-KO Ramos cells; WT, Ramos wild-type cells.

Article Snippet: 0.6 × 106 or 1 × 106 cells were incubated in 1 ml RPMI (Thermo Fisher Scientific) + 1% FBS (Biochrom AG) + 15 μl Indo-1 solution at 37°C for 45 min.

Techniques: Staining, Incubation, Cell Culture

Journal: Cancers

Article Title: Plasmacytoid Dendritic Cell Impairment in Metastatic Melanoma by Lactic Acidosis

doi: 10.3390/cancers12082085

Figure Lengend Snippet: In vitro lactic acidosis affects IFN-α production by pDCs. pDCs purified from buffy coats of HD were cultured in RPMI 1640 medium supplemented with 10% FBS and IL-3 plus lactic acid (10 mM; 15 mM; 20 mM) ( n = 7; ( A )) or hydrochloric acid (pH = 7.4; 7.0; 6.5; 6.0; 5.5) ( n = 7; ( B )) for 24 h. pDCs were stimulated with R848 for 2 h. Intracellular IFN-α was analyzed by flow cytometry. Aligned dot plot graphs show the percentages of IFN-α + pDCs evaluated on BDCA-2 + /CD123 + cells. Bars represent the mean of biological replicates. The statistical significance was calculated by two-sample paired sign test. * p

Article Snippet: In addition, pDCs and T lymphocytes were cultured in RPMI 1640 medium supplemented with 10% FBS (Biochrom GmbH, Holliston, MA, USA), titrated to pH ≈ 7.4, 7.0, 6.5, 6.0, and 5.5 using HCl.

Techniques: In Vitro, Purification, Cell Culture, Flow Cytometry

Journal: Cancers

Article Title: Plasmacytoid Dendritic Cell Impairment in Metastatic Melanoma by Lactic Acidosis

doi: 10.3390/cancers12082085

Figure Lengend Snippet: Lactic acidosis affects the viability of pDCs and T cells. pDCs and T cells purified from buffy coats of HD were cultured in RPMI 1640 medium supplemented with 10% FBS plus lactic Acid (10 mM; 15 mM; 20 mM) ( n = 7 ( A – C ); n = 6, ( D – F )) or hydrochloric acid (pH = 7.4; 7.0; 6.5; 6.0; 5.5 ( n = 6 ( G – I ); n = 7, ( J – L )) for 24 h. IL-3 was added to pDCs’ culture. The cellular viability was analyzed by annexin V/SYTOX AADvanced staining in flow cytometry. Aligned dot plot graphs show the percentages of dead ( A , D , G , J ), early apoptotic ( B , E , H , K ), and late apoptotic or necrotic cells ( C , F , I , L ). Bars represent the mean of biological replicates. The statistical significance was calculated by two-sample paired sign test. * p

Article Snippet: In addition, pDCs and T lymphocytes were cultured in RPMI 1640 medium supplemented with 10% FBS (Biochrom GmbH, Holliston, MA, USA), titrated to pH ≈ 7.4, 7.0, 6.5, 6.0, and 5.5 using HCl.

Techniques: Purification, Cell Culture, Staining, Flow Cytometry

Journal: Cancers

Article Title: Plasmacytoid Dendritic Cell Impairment in Metastatic Melanoma by Lactic Acidosis

doi: 10.3390/cancers12082085

Figure Lengend Snippet: Frequency of interferon alpha (IFN-α) and CXCL10-producing plasmacytoid dendritic cells (pDCs) in chemo-naïve MM patients and HD. Representative dot plots of R848-stimulated IFN-α + and CXCL10 + pDC subsets obtained from HD and MM patients are shown ( A ). PBMCs were isolated from peripheral blood of HD ( n = 25) and MM patients ( n = 29). Total PBMCs were cultured in RPMI 1640 medium supplemented with 10% FBS and IL-3 and stimulated with R848 or IMQ for 2 h ( B , D ) and 6 h ( C , E ), and with CpG-ODN 2216 for 6 h ( B – E ). IFN-α ( B , D ) and CXCL10 ( C , E ) were analyzed by intracellular flow cytometry staining. Scatter dot plot graphs illustrate the percentages of positive pDCs evaluated on BDCA-2 + /CD123 + cells. Subgroup analysis of the MM cohort illustrating the frequency of IFN-α + and CXCL10 + pDCs in M1a-c categories ( D , E ). Median and IQR are shown in ( B , C ). Mean and SD are shown in ( D , E ). The statistical significance was calculated by Wilcoxon–Mann–Whitney test ( B , C ) and by a Student’s t -test ( D , E ). * p

Article Snippet: In addition, pDCs and T lymphocytes were cultured in RPMI 1640 medium supplemented with 10% FBS (Biochrom GmbH, Holliston, MA, USA), titrated to pH ≈ 7.4, 7.0, 6.5, 6.0, and 5.5 using HCl.

Techniques: Isolation, Cell Culture, Flow Cytometry, Staining, MANN-WHITNEY

Journal: Cell

Article Title: Regulation of TORC1 in response to amino acid starvation via lysosomal recruitment of TSC2

doi: 10.1016/j.cell.2014.01.024

Figure Lengend Snippet: Amino acid starvation causes TSC2 lysosomal localization in a Rag-dependent manner (A,C) TSC2 accumulates on lysosomes upon amino acid deprivation. Confocal micrograph of HEK293FT cells (A) or MEFs (C) treated +/− all amino acids in the presence of dialyzed FBS for the indicated times, stained for TSC2 (green) and the lysosomal marker LAMP2 (red). LAMP2 aggregates do not show TSC2 accumulation in the +aa condition. (B) TSC2 quickly relocalizes away from lysosomes upon amino acid readdition. HEK293FT cells, starved for amino acids in the presence of dialyzed serum for 4h, then re-supplied with amino acid-containing media for 5 minutes. Cells stained and imaged as in (A).

Article Snippet: HEK293FT cells (Invitrogen) and mouse embryonic fibroblasts were cultured in high glucose DMEM, supplemented with 10% FBS (Biochrom), except PTEN−/− MEFS which were also supplemented with 2mM Glutamine.

Techniques: Staining, Marker

Journal: Cell

Article Title: Regulation of TORC1 in response to amino acid starvation via lysosomal recruitment of TSC2

doi: 10.1016/j.cell.2014.01.024

Figure Lengend Snippet: Lysosomal recruitment of TSC2 depends on the Rag proteins (A-A’) Lysosomal localization of TSC2 upon amino acid removal requires the LAMTOR complex. Confocal micrograph of p14/LAMTOR2 knock-out MEFs (A), or control p14 knock-out MEFs reconstituted to express an EGFP-p14 fusion (A’), treated +/− amino acids for 1h in the presence of dialyzed FBS. Lysosomes marked either with anti-LAMP2 antibody or with the lysosomaly localized EGFP-p14. (B) Lysosomal localization of TSC2 upon amino acid removal requires the Rag proteins. MEFs transfected with control siRNA against Renilla luciferase or siRNAs targeting the various Rag proteins, treated +/− amino acids for 1h in the presence of dialyzed FBS. Cells stained and imaged as in (A). (C) Lysosomal localization of TSC2 upon amino acid removal requires that the Rag proteins change to the inactive conformation. MEFs transfected with control siRNA or siRNAs targeting components of the GATOR1 complex DEPDC5, NPRL2, or NPRL3 required for the Rag proteins to become inactive upon amino acid removal. Cells stained and imaged as in (A).

Article Snippet: HEK293FT cells (Invitrogen) and mouse embryonic fibroblasts were cultured in high glucose DMEM, supplemented with 10% FBS (Biochrom), except PTEN−/− MEFS which were also supplemented with 2mM Glutamine.

Techniques: Knock-Out, Transfection, Luciferase, Staining

Journal: Cell

Article Title: Regulation of TORC1 in response to amino acid starvation via lysosomal recruitment of TSC2

doi: 10.1016/j.cell.2014.01.024

Figure Lengend Snippet: TSC2 is required for mTOR to localize away from lysosomes upon amino acid removal in a Rheb dependent manner (A-B) mTOR is released from lysosomes upon amino acid removal in control (A) but not TSC2-null MEFs (A’). This is rescued by re-expressing TSC2 + EGFP (to mark transfected cells) but not EGFP alone in the TSC-null MEFs (B). Note that the cell expressing TSC2 and GFP no longer has mTOR accumulated on lysosomes, whereas the surrounding, non-transfected cells retain lysosomally localized mTOR. (C) Defective release of mTOR from lysosomes upon amino acid withdrawal in TSC2-null MEFs is rescued by knocking down Rheb. TSC2-null MEFs transfected with either Rheb siRNAs (upper panels) or control siRNAs (lower panels) treated 1h with +/− amino acid containing medium in the presence of dialyzed FBS.

Article Snippet: HEK293FT cells (Invitrogen) and mouse embryonic fibroblasts were cultured in high glucose DMEM, supplemented with 10% FBS (Biochrom), except PTEN−/− MEFS which were also supplemented with 2mM Glutamine.

Techniques: Expressing, Transfection