Structured Review

Cellgro fetal bovine serum fbs
GSP induces significantly less apoptosis in p53-deficient (p53 -/- ) cells compared to p53 wild-type (p53 +/+ ) cells. Both p53 +/+ and p53 -/- fibroblast cells were cultured under identical conditions as was done in JB6 C141 cells except that the cells were treated with 15% <t>FBS</t> in <t>DMEM.</t> Cells were starved in 0.5% FBS/DMEM overnight and thereafter treated with GSP in serum-containing media for another 24 hours. Morphologic changes in p53 +/+ and p53 -/- fibroblast cells undergoing apoptosis were observed under fluorescence microscopy. Cells in panels A and G were not treated with GSP (controls), whereas increasing concentrations of GSP were added to cultures for 24 hours after overnight serum starvation in panels B–E and H–K by 20, 40, 60, and 80 µg/ml, respectively. The percentage of apoptotic cells from panel A to panel E is summarized in panel F, and the percentage of apoptotic cells from panel G to panel K is summarized in panel L. The experiment was repeated at least two times and the average percentage of apoptotic cells ± SD for each treatment group is indicated. At least 200 cells were counted in a blinded manner to score the percentage of apoptosis in each treatment group. *P
Fetal Bovine Serum Fbs, supplied by Cellgro, used in various techniques. Bioz Stars score: 93/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/fetal bovine serum fbs/product/Cellgro
Average 93 stars, based on 18 article reviews
Price from $9.99 to $1999.99
fetal bovine serum fbs - by Bioz Stars, 2020-07
93/100 stars

Images

1) Product Images from "Grape Seed Proanthocyanidins Induce Apoptosis through p53, Bax, and Caspase 3 Pathways 1"

Article Title: Grape Seed Proanthocyanidins Induce Apoptosis through p53, Bax, and Caspase 3 Pathways 1

Journal: Neoplasia (New York, N.Y.)

doi:

GSP induces significantly less apoptosis in p53-deficient (p53 -/- ) cells compared to p53 wild-type (p53 +/+ ) cells. Both p53 +/+ and p53 -/- fibroblast cells were cultured under identical conditions as was done in JB6 C141 cells except that the cells were treated with 15% FBS in DMEM. Cells were starved in 0.5% FBS/DMEM overnight and thereafter treated with GSP in serum-containing media for another 24 hours. Morphologic changes in p53 +/+ and p53 -/- fibroblast cells undergoing apoptosis were observed under fluorescence microscopy. Cells in panels A and G were not treated with GSP (controls), whereas increasing concentrations of GSP were added to cultures for 24 hours after overnight serum starvation in panels B–E and H–K by 20, 40, 60, and 80 µg/ml, respectively. The percentage of apoptotic cells from panel A to panel E is summarized in panel F, and the percentage of apoptotic cells from panel G to panel K is summarized in panel L. The experiment was repeated at least two times and the average percentage of apoptotic cells ± SD for each treatment group is indicated. At least 200 cells were counted in a blinded manner to score the percentage of apoptosis in each treatment group. *P
Figure Legend Snippet: GSP induces significantly less apoptosis in p53-deficient (p53 -/- ) cells compared to p53 wild-type (p53 +/+ ) cells. Both p53 +/+ and p53 -/- fibroblast cells were cultured under identical conditions as was done in JB6 C141 cells except that the cells were treated with 15% FBS in DMEM. Cells were starved in 0.5% FBS/DMEM overnight and thereafter treated with GSP in serum-containing media for another 24 hours. Morphologic changes in p53 +/+ and p53 -/- fibroblast cells undergoing apoptosis were observed under fluorescence microscopy. Cells in panels A and G were not treated with GSP (controls), whereas increasing concentrations of GSP were added to cultures for 24 hours after overnight serum starvation in panels B–E and H–K by 20, 40, 60, and 80 µg/ml, respectively. The percentage of apoptotic cells from panel A to panel E is summarized in panel F, and the percentage of apoptotic cells from panel G to panel K is summarized in panel L. The experiment was repeated at least two times and the average percentage of apoptotic cells ± SD for each treatment group is indicated. At least 200 cells were counted in a blinded manner to score the percentage of apoptosis in each treatment group. *P

Techniques Used: Cell Culture, Fluorescence, Microscopy

Treatment of GSP decreases the expression of the antiapoptotic proteins, Bcl-2 and Bcl-xl, and increases the expression of the pro-apoptotic protein, Bax, in JB6 C141 cells. JB6 C141 cells were starved in 0.5% FBS/DMEM overnight and then treated with GSP (20–80 µg/ml) in serum-containing media for another 24 hours. Cell lysates were prepared, and the expression of the proteins was determined by Western blot analysis using the corresponding antibodies, as detailed in the Materials and Methods section. Relative intensity of bands in each panel was determined. GSP treatment to JB6 C141 cells decreased the expressions of the antiapoptotic proteins, Bcl-2 (panel A) and Bcl-xl (panel D), whereas the expression of the pro-apoptotic protein, Bax, was increased (panel B). A representative blot is shown from three independent experiments with identical results. The ratio of Bax and Bcl-2 protein expression was determined from three separate experiments by comparing the relative intensities of protein bands and shown as a mean ± SD (panel C). β-Actin was used as an internal control to monitor equal protein loading and transfer of proteins from the gel to the membranes after stripping them and reprobing them with the actin antibody.
Figure Legend Snippet: Treatment of GSP decreases the expression of the antiapoptotic proteins, Bcl-2 and Bcl-xl, and increases the expression of the pro-apoptotic protein, Bax, in JB6 C141 cells. JB6 C141 cells were starved in 0.5% FBS/DMEM overnight and then treated with GSP (20–80 µg/ml) in serum-containing media for another 24 hours. Cell lysates were prepared, and the expression of the proteins was determined by Western blot analysis using the corresponding antibodies, as detailed in the Materials and Methods section. Relative intensity of bands in each panel was determined. GSP treatment to JB6 C141 cells decreased the expressions of the antiapoptotic proteins, Bcl-2 (panel A) and Bcl-xl (panel D), whereas the expression of the pro-apoptotic protein, Bax, was increased (panel B). A representative blot is shown from three independent experiments with identical results. The ratio of Bax and Bcl-2 protein expression was determined from three separate experiments by comparing the relative intensities of protein bands and shown as a mean ± SD (panel C). β-Actin was used as an internal control to monitor equal protein loading and transfer of proteins from the gel to the membranes after stripping them and reprobing them with the actin antibody.

Techniques Used: Expressing, Western Blot, Stripping Membranes

2) Product Images from "Mycobacterium avium Biofilm Attenuates Mononuclear Phagocyte Function by Triggering Hyperstimulation and Apoptosis during Early Infection"

Article Title: Mycobacterium avium Biofilm Attenuates Mononuclear Phagocyte Function by Triggering Hyperstimulation and Apoptosis during Early Infection

Journal: Infection and Immunity

doi: 10.1128/IAI.00820-13

THP-1 cells are stimulated during exposure to M. avium subsp. hominissuis biofilms. (A) THP-1 cells were added on top of M. avium subsp. hominissuis strain A5 and 104 14-day-old biofilms (equal inoculum levels), and supernatant was collected at 0.5, 1, 2, 4, 24, and 48 h. TNF-α ELISA was carried out for each time point for each strain. (B) Filter-sterilized supernatants from 14-day-old biofilms were added at a 10% (vol/vol) concentration in RPMI 1640 plus 10% FBS with THP-1 cells for 4 h. Culture supernatant was then collected, and TNF-α ELISA was performed. Abbreviations: NS, not stimulated; 104 S, M. avium subsp. hominissuis 104 supernatant; A5 S, M. avium subsp. hominissuis A5 supernatant. (C) UV-killed M. avium subsp. hominissuis A5 biofilms (A5 BF UV) were compared side by side with non-UV-treated biofilms (A5 BF) and planktonic bacteria that were immobilized on the bottom surface of the plate via centrifugation (A5 plank) for TNF-α production by THP-1 cells 4 h after being placed on top of the bacteria. (D) THP-1 cells were placed on top of M. avium subsp. hominissuis A5 biofilm, and O 2 − was assessed spectrophotometrically as described in Materials and Methods. (E) THP-1 cells were placed on top of M. avium subsp. hominissuis A5 biofilm, and nitric oxide (NO) was assessed using the Griess reagent system. Abbreviations for panels D and E: neg, negative control for respective assay; THP-1, THP-1 cells placed in empty wells with no bacteria present; THP-1 A5, THP-1 cells placed in wells containing M. avium subsp. hominissuis A5 biofilm. Bars represent means ± standard deviations. *, P
Figure Legend Snippet: THP-1 cells are stimulated during exposure to M. avium subsp. hominissuis biofilms. (A) THP-1 cells were added on top of M. avium subsp. hominissuis strain A5 and 104 14-day-old biofilms (equal inoculum levels), and supernatant was collected at 0.5, 1, 2, 4, 24, and 48 h. TNF-α ELISA was carried out for each time point for each strain. (B) Filter-sterilized supernatants from 14-day-old biofilms were added at a 10% (vol/vol) concentration in RPMI 1640 plus 10% FBS with THP-1 cells for 4 h. Culture supernatant was then collected, and TNF-α ELISA was performed. Abbreviations: NS, not stimulated; 104 S, M. avium subsp. hominissuis 104 supernatant; A5 S, M. avium subsp. hominissuis A5 supernatant. (C) UV-killed M. avium subsp. hominissuis A5 biofilms (A5 BF UV) were compared side by side with non-UV-treated biofilms (A5 BF) and planktonic bacteria that were immobilized on the bottom surface of the plate via centrifugation (A5 plank) for TNF-α production by THP-1 cells 4 h after being placed on top of the bacteria. (D) THP-1 cells were placed on top of M. avium subsp. hominissuis A5 biofilm, and O 2 − was assessed spectrophotometrically as described in Materials and Methods. (E) THP-1 cells were placed on top of M. avium subsp. hominissuis A5 biofilm, and nitric oxide (NO) was assessed using the Griess reagent system. Abbreviations for panels D and E: neg, negative control for respective assay; THP-1, THP-1 cells placed in empty wells with no bacteria present; THP-1 A5, THP-1 cells placed in wells containing M. avium subsp. hominissuis A5 biofilm. Bars represent means ± standard deviations. *, P

Techniques Used: Enzyme-linked Immunosorbent Assay, Concentration Assay, Centrifugation, Negative Control

3) Product Images from "Ursolic acid protects monocytes against metabolic stress-induced priming and dysfunction by preventing the induction of Nox4"

Article Title: Ursolic acid protects monocytes against metabolic stress-induced priming and dysfunction by preventing the induction of Nox4

Journal: Redox Biology

doi: 10.1016/j.redox.2014.01.003

UA reduces actin- and total- S -glutathionylation induced by metabolic stress. THP-1 monocytes in RPMI 1640 medium (5 mM glucose, 10% FBS) were treated with 0.3, 1, 3, 10 µM UA or vehicle. HG (20 mM glucose) plus native LDL (100 µg/ml) was present for 20 h where indicated. Cells were lysed in the lysis buffer containing 10 mM NEM. Actin- and protein- S -glutathionylation was assessed by Western blot analysis using the anti-glutathione antibody. Western Blot data for actin- S -glutathionylation is summarized in A–C. (A) A representative Western Blot is shown. (B) Quantitation by Western blot analysis assessed using an anti-glutathione antibody is shown of actin-S-glutathionylation in response to increasing doses of UA. n =4, mean±SE. # versus 100% actin- S -glutathionylation, P =0.004 (1 µM), P =0.003 (3 µM), P ≤0.001 (10 µM). (C) Quantitative data for actin- S -glutathionylation and the effects of 3 µM UA. Data is represented as fold change induced by HG+LDL (red bar) and HG+LDL+ 3 µM UA (green bar) versus unprimed control cells (white bar). n =3, mean±SE; ⁎ versus Control, P =0.006, # versus HG+LDL, P =0.022. (D) Total protein- S -glutathionylation was determined by Western blot and the density of the entire lane was measured and normalized to actin. Total protein- S -glutathionylation is represented as fold change induced by HG+LDL (red bar) and HG+LDL+3 µM UA (green bar) versus unprimed control cells (white bar). n =4, mean±SE, ⁎ versus control, P
Figure Legend Snippet: UA reduces actin- and total- S -glutathionylation induced by metabolic stress. THP-1 monocytes in RPMI 1640 medium (5 mM glucose, 10% FBS) were treated with 0.3, 1, 3, 10 µM UA or vehicle. HG (20 mM glucose) plus native LDL (100 µg/ml) was present for 20 h where indicated. Cells were lysed in the lysis buffer containing 10 mM NEM. Actin- and protein- S -glutathionylation was assessed by Western blot analysis using the anti-glutathione antibody. Western Blot data for actin- S -glutathionylation is summarized in A–C. (A) A representative Western Blot is shown. (B) Quantitation by Western blot analysis assessed using an anti-glutathione antibody is shown of actin-S-glutathionylation in response to increasing doses of UA. n =4, mean±SE. # versus 100% actin- S -glutathionylation, P =0.004 (1 µM), P =0.003 (3 µM), P ≤0.001 (10 µM). (C) Quantitative data for actin- S -glutathionylation and the effects of 3 µM UA. Data is represented as fold change induced by HG+LDL (red bar) and HG+LDL+ 3 µM UA (green bar) versus unprimed control cells (white bar). n =3, mean±SE; ⁎ versus Control, P =0.006, # versus HG+LDL, P =0.022. (D) Total protein- S -glutathionylation was determined by Western blot and the density of the entire lane was measured and normalized to actin. Total protein- S -glutathionylation is represented as fold change induced by HG+LDL (red bar) and HG+LDL+3 µM UA (green bar) versus unprimed control cells (white bar). n =4, mean±SE, ⁎ versus control, P

Techniques Used: Lysis, Western Blot, Quantitation Assay

UA prevents Nox4 protein induction by metabolic stress. (A) THP-1 monocytes cultured in RPMI 1640 medium (5 mM glucose, 10% FBS) were treated with 3 µM UA, 3 µM OA, or vehicle for 1 h prior to the addition of glucose (20 mM HG) plus native LDL (100 µg/ml) for an additional 20 h. Nox4 protein expression was determined by Western blot analysis as described under “Material and methods” section. Data was normalized to actin levels. Results are shown as mean±SE of 3–4 independent experiments. ⁎ versus unprimed control (open bars, no metabolic stress), P
Figure Legend Snippet: UA prevents Nox4 protein induction by metabolic stress. (A) THP-1 monocytes cultured in RPMI 1640 medium (5 mM glucose, 10% FBS) were treated with 3 µM UA, 3 µM OA, or vehicle for 1 h prior to the addition of glucose (20 mM HG) plus native LDL (100 µg/ml) for an additional 20 h. Nox4 protein expression was determined by Western blot analysis as described under “Material and methods” section. Data was normalized to actin levels. Results are shown as mean±SE of 3–4 independent experiments. ⁎ versus unprimed control (open bars, no metabolic stress), P

Techniques Used: Cell Culture, Expressing, Western Blot

UA attenuates metabolic stress-induced acceleration of monocyte chemotaxis in response to MCP-1. (A) THP-1 cells cultured in RPMI 1640 medium (5 mM glucose, 10% FBS) were treated for 20 h with HG (20 mM d -glucose) and native LDL (100 µg/ml) in the presence of 0, 0.3, 1.0, 3.0 or 10 µM UA or vehicle (DMSO). The supernatant was removed and cells were resuspended in 0.1% FBS-containing RPMI medium. Cells were then transferred into a multi-well Boyden chamber and stimulated with 2 nM MCP-1 for 2 h. Migrated cells were counted in 4 high-power fields (HPF) per well, 4 wells for each condition. Data were normalized to the accelerating effect of metabolic stress on chemotaxis (“100%”), i.e., values obtained for HG+LDL-primed THP-1 monocytes stimulated with MCP-1 minus values obtained from unprimed THP-1 monocytes stimulated with MCP-1 (“0%”;dotted line). Results are shown as mean from 5 independent experiments±SE; #versus 100% acceleration, P =0.038 (0.3 µM), P =0.002 (1, 3 µM), P
Figure Legend Snippet: UA attenuates metabolic stress-induced acceleration of monocyte chemotaxis in response to MCP-1. (A) THP-1 cells cultured in RPMI 1640 medium (5 mM glucose, 10% FBS) were treated for 20 h with HG (20 mM d -glucose) and native LDL (100 µg/ml) in the presence of 0, 0.3, 1.0, 3.0 or 10 µM UA or vehicle (DMSO). The supernatant was removed and cells were resuspended in 0.1% FBS-containing RPMI medium. Cells were then transferred into a multi-well Boyden chamber and stimulated with 2 nM MCP-1 for 2 h. Migrated cells were counted in 4 high-power fields (HPF) per well, 4 wells for each condition. Data were normalized to the accelerating effect of metabolic stress on chemotaxis (“100%”), i.e., values obtained for HG+LDL-primed THP-1 monocytes stimulated with MCP-1 minus values obtained from unprimed THP-1 monocytes stimulated with MCP-1 (“0%”;dotted line). Results are shown as mean from 5 independent experiments±SE; #versus 100% acceleration, P =0.038 (0.3 µM), P =0.002 (1, 3 µM), P

Techniques Used: Chemotaxis Assay, Cell Culture

4) Product Images from "Research Upregulation of CD23 (Fc?RII) Expression in Human Airway Smooth Muscle Cells (huASMC) in Response to IL-4, GM-CSF, and IL-4/GM-CSF"

Article Title: Research Upregulation of CD23 (Fc?RII) Expression in Human Airway Smooth Muscle Cells (huASMC) in Response to IL-4, GM-CSF, and IL-4/GM-CSF

Journal: Clinical and molecular allergy : CMA

doi: 10.1186/1476-7961-3-6

Upregulation of CD23 by IL-4, GM-CSF and IL-4/GM-CSF . Alpha-smooth muscle isoactin positive Human ASMC (Clonetics) in T-75 flasks were starved for 24 h in 0.1% FBS containing medium M199. The cells were then stimulated with IL-4 (0.5 nM)/GM-CSF (0.4 nM) for 24 h. The FACS analysis showed that the smaller cells passed through Gate A and larger cells passed through Gate D. Background noise was eliminated using the BSA-stimulated control cells that were labeled with PE-anti-CD23 (EBVCS), represented by C in Gate A, and F in Gate D. The FACS results of a representative experiment showed 66% of the larger cells (Gate D) had an increase in cell expression of CD23 when compared to the controls.
Figure Legend Snippet: Upregulation of CD23 by IL-4, GM-CSF and IL-4/GM-CSF . Alpha-smooth muscle isoactin positive Human ASMC (Clonetics) in T-75 flasks were starved for 24 h in 0.1% FBS containing medium M199. The cells were then stimulated with IL-4 (0.5 nM)/GM-CSF (0.4 nM) for 24 h. The FACS analysis showed that the smaller cells passed through Gate A and larger cells passed through Gate D. Background noise was eliminated using the BSA-stimulated control cells that were labeled with PE-anti-CD23 (EBVCS), represented by C in Gate A, and F in Gate D. The FACS results of a representative experiment showed 66% of the larger cells (Gate D) had an increase in cell expression of CD23 when compared to the controls.

Techniques Used: FACS, Labeling, Expressing

Western blot analysis of CD23 after stimulation of IL-4, GM-CSF, IL-4/GM-CSF . Alpha-smooth muscle isoactin positive huASMC (Clonetics) in T-75 flasks were starved for 24 h in 0.1% FBS containing medium M199. The cells were then stimulated with BSA (1 μg/ml) (vehicle control), IL-4 (0.5 nM), GM-CSF (0.4 nM), or IL-4/GM-CSF (0.5 nM/0.4 nM) for 24 h. The cell lysates in RIPA buffer were subjected to western blot analysis for CD23. Mouse anti-human CD23 monoclonal antibody (clone M-L233, BD Biosciences, 1 μg/5 ml) was used as the primary antibody and anti-mouse horseradish peroxidase linked antibody as the secondary antibody (Amersham). The immunoreactive protein bands were detected by enhanced chemiluminescence light (ECL) (Amersham). Paxillin mouse monoclonal IgG 1 (Transduction Laboratories) was used as an irrelevant isotype control.
Figure Legend Snippet: Western blot analysis of CD23 after stimulation of IL-4, GM-CSF, IL-4/GM-CSF . Alpha-smooth muscle isoactin positive huASMC (Clonetics) in T-75 flasks were starved for 24 h in 0.1% FBS containing medium M199. The cells were then stimulated with BSA (1 μg/ml) (vehicle control), IL-4 (0.5 nM), GM-CSF (0.4 nM), or IL-4/GM-CSF (0.5 nM/0.4 nM) for 24 h. The cell lysates in RIPA buffer were subjected to western blot analysis for CD23. Mouse anti-human CD23 monoclonal antibody (clone M-L233, BD Biosciences, 1 μg/5 ml) was used as the primary antibody and anti-mouse horseradish peroxidase linked antibody as the secondary antibody (Amersham). The immunoreactive protein bands were detected by enhanced chemiluminescence light (ECL) (Amersham). Paxillin mouse monoclonal IgG 1 (Transduction Laboratories) was used as an irrelevant isotype control.

Techniques Used: Western Blot, Transduction

Expression of CD23 in response to IL-4/GM-CSF is accompanied by changes in huASMC morphology . Alpha-smooth muscle isoactin positive huASMC (Clonetics) in T-75 flasks were starved for 24 h in 0.1% FBS containing medium M199. The cells were then stimulated with either BSA or IL-4 (0.5 nM)/GM-CSF (0.4 nM) for 24 h, and stained with either anti-smooth muscle-isoactin (A B) or anti-CD23 antibody (C D). Those cells stimulated with the combination of IL-4/GM-CSF demonstrated CD23 expression (D) and changes in cell morphology including depolymerization of isoactin fibers, cell spreading, and membrane ruffling (B). Cells stimulated with BSA (vehicle for IL-4/GM-CSF) alone did not increase the expression of CD23 (C) nor changes in phenotype (A). These findings were confirmed by three independent observers.
Figure Legend Snippet: Expression of CD23 in response to IL-4/GM-CSF is accompanied by changes in huASMC morphology . Alpha-smooth muscle isoactin positive huASMC (Clonetics) in T-75 flasks were starved for 24 h in 0.1% FBS containing medium M199. The cells were then stimulated with either BSA or IL-4 (0.5 nM)/GM-CSF (0.4 nM) for 24 h, and stained with either anti-smooth muscle-isoactin (A B) or anti-CD23 antibody (C D). Those cells stimulated with the combination of IL-4/GM-CSF demonstrated CD23 expression (D) and changes in cell morphology including depolymerization of isoactin fibers, cell spreading, and membrane ruffling (B). Cells stimulated with BSA (vehicle for IL-4/GM-CSF) alone did not increase the expression of CD23 (C) nor changes in phenotype (A). These findings were confirmed by three independent observers.

Techniques Used: Expressing, Staining

Upregulation of IL-2Rγc Expression in huASMC by IL-4 and IL-4/GM-CSF . Alpha-smooth muscle isoactin positive Human ASMC (Clonetics) in T-75 flasks were starved for 24 h in 0.1% FBS containing medium M199. The cells were then either stimulated with BSA (vehicle) (1 μg/ml), IL-4 (0.5 nM), GM-CSF (0.4 nM), or IL-4/GM-CSF (0.5 nM/0.4 nM) for 24 hours. The IL-4 and IL-4/GM-CSF stimulated cells had increased IL-2Rγc expression compared to the BSA (vehicle) group. Fibronectin polyclonal rabbit antibody (Sigma) (1:250) was used as an irrelevant isotype control.
Figure Legend Snippet: Upregulation of IL-2Rγc Expression in huASMC by IL-4 and IL-4/GM-CSF . Alpha-smooth muscle isoactin positive Human ASMC (Clonetics) in T-75 flasks were starved for 24 h in 0.1% FBS containing medium M199. The cells were then either stimulated with BSA (vehicle) (1 μg/ml), IL-4 (0.5 nM), GM-CSF (0.4 nM), or IL-4/GM-CSF (0.5 nM/0.4 nM) for 24 hours. The IL-4 and IL-4/GM-CSF stimulated cells had increased IL-2Rγc expression compared to the BSA (vehicle) group. Fibronectin polyclonal rabbit antibody (Sigma) (1:250) was used as an irrelevant isotype control.

Techniques Used: Expressing

Upregulation of CD23 by IL-4 and GM-CSF . Dose-ranging studies were performed to determine the optimum concentrations of IL-4 and GM-CSF. Alpha-smooth muscle isoactin positive Human ASMC (Clonetics) in T-75 flasks were starved for 24 h in 0.1% FBS containing medium M199. The cells were then stimulated with BSA (1 μg/ml), IL-4 (0.125. 0.25, 0.5, or 1.0 nM) or GM-CSF (0.1, 0.2, 0.4, or 0.8 nM) for 24 h. The cell lysates in RIPA buffer were subjected to western blot analysis for CD23. Mouse anti-human CD23 monoclonal antibody (clone M-L233, BD Biosciences, 1 μg/5 ml) was used as the primary antibody and anti-mouse horseradish peroxidase linked antibody as the secondary antibody (Amersham). The immunoreactive protein bands were detected by enhanced chemiluminescence light (ECL) (Amersham).
Figure Legend Snippet: Upregulation of CD23 by IL-4 and GM-CSF . Dose-ranging studies were performed to determine the optimum concentrations of IL-4 and GM-CSF. Alpha-smooth muscle isoactin positive Human ASMC (Clonetics) in T-75 flasks were starved for 24 h in 0.1% FBS containing medium M199. The cells were then stimulated with BSA (1 μg/ml), IL-4 (0.125. 0.25, 0.5, or 1.0 nM) or GM-CSF (0.1, 0.2, 0.4, or 0.8 nM) for 24 h. The cell lysates in RIPA buffer were subjected to western blot analysis for CD23. Mouse anti-human CD23 monoclonal antibody (clone M-L233, BD Biosciences, 1 μg/5 ml) was used as the primary antibody and anti-mouse horseradish peroxidase linked antibody as the secondary antibody (Amersham). The immunoreactive protein bands were detected by enhanced chemiluminescence light (ECL) (Amersham).

Techniques Used: Western Blot

5) Product Images from "Metabolic assessment of a novel chronic myelogenous leukemic cell line and an imatinib resistant subline by 1H NMR spectroscopy"

Article Title: Metabolic assessment of a novel chronic myelogenous leukemic cell line and an imatinib resistant subline by 1H NMR spectroscopy

Journal: Metabolomics

doi: 10.1007/s11306-010-0204-0

1 H NMR spectra from MyL and MyL-R cells extracts. MyL ( a ) and MyL-R ( b ) cells were expanded and maintained in T175 flasks containing RPMI culture medium with 10% FBS in the absence of imatinib. The expanded cells were collected by low speed centrifugation, counted and then plated at a density 5.0 × 10 6 cells/ml in 20 ml of fresh medium. After incubation for 2 h at 37°C, the cells were collected, washed three times with cold PBS, and extracted as described in Sect. 2 . Cell extracts were evaporated to dryness followed by suspension in D 2 O containing TSP (internal standard), and analyzed by 1 H NMR. Spectra are representative for each cell type and highlight major metabolites identified. Lac lactate, m-Ins myo-inositol, Gly glycine, Glm glutamine, PCho phosphocholine, Cho choline, Cr creatine, GSH glutathione, Suc succinate, Pro proline, Ace acetate, Ala alanine, Val valine, Ileu isoleucine, Leu leucine, NAA n -acetylaspartate
Figure Legend Snippet: 1 H NMR spectra from MyL and MyL-R cells extracts. MyL ( a ) and MyL-R ( b ) cells were expanded and maintained in T175 flasks containing RPMI culture medium with 10% FBS in the absence of imatinib. The expanded cells were collected by low speed centrifugation, counted and then plated at a density 5.0 × 10 6 cells/ml in 20 ml of fresh medium. After incubation for 2 h at 37°C, the cells were collected, washed three times with cold PBS, and extracted as described in Sect. 2 . Cell extracts were evaporated to dryness followed by suspension in D 2 O containing TSP (internal standard), and analyzed by 1 H NMR. Spectra are representative for each cell type and highlight major metabolites identified. Lac lactate, m-Ins myo-inositol, Gly glycine, Glm glutamine, PCho phosphocholine, Cho choline, Cr creatine, GSH glutathione, Suc succinate, Pro proline, Ace acetate, Ala alanine, Val valine, Ileu isoleucine, Leu leucine, NAA n -acetylaspartate

Techniques Used: Nuclear Magnetic Resonance, Centrifugation, Incubation

Related Articles

Flow Cytometry:

Article Title: Research Upregulation of CD23 (Fc?RII) Expression in Human Airway Smooth Muscle Cells (huASMC) in Response to IL-4, GM-CSF, and IL-4/GM-CSF
Article Snippet: .. Cell culture and flow cytometry Alpha-smooth muscle isoactin positive Human ASMC (Cambrex, Walkersville, MD) in T-75 flasks were starved for 24 hours in 0.1% (vol/vol) fetal bovine serum (FBS) containing medium M199 (Cellgro, Herndon, VA) supplemented with 1% (vol/vol) antibiotic/antimycotic solution (Sigma Chemical Co., St Louis, MO). .. The cells were then stimulated with either vehicle (bovine serum albumin, BSA, vehicle for cytokines (1 mg/ml), in M199; ethanol (EtOH), vehicle for LTD4 (6% final concentration) and PGD2 (0.001–0.01% final concentration); M199, vehicle for tryptase), an individual mediator, or a mediator in combination with GM-CSF at their optimum concentrations for 24 hours.

Isolation:

Article Title: Ursolic acid protects monocytes against metabolic stress-induced priming and dysfunction by preventing the induction of Nox4
Article Snippet: .. Briefly, human THP-1 monocytes (ATCC, Manassas, VA) at 1–2×106 cells/ml were cultured at 37 °C for 20 h in RPMI-1640 (Hyclone and Cellgro® ) containing, 10% fetal bovine serum (FBS), 5.5 mM d -glucose, 2% Glutamax, 1% sodium pyruvate (Cellgro® ), 1% penicillin/streptomycin (Cellgro® ), 1% HEPES, 0.1% β-2-mercaptoethanol, and supplemented with either phosphate buffered saline (PBS) or freshly isolated native human LDL (100 µg/ml in PBS) plus d -glucose (high glucose, 20 mM). l -glucose does not increase monocyte priming . .. For selected experiments, peritoneal macrophages were collected from C57BL/6 mice by peritoneal lavage and purified by negative selection using antibody-coated magnetic beads (Dynabeads® mouse pan B (B220) and Dynabeads® mouse pan T (Thy 1.2)).

Cytometry:

Article Title: Research Upregulation of CD23 (Fc?RII) Expression in Human Airway Smooth Muscle Cells (huASMC) in Response to IL-4, GM-CSF, and IL-4/GM-CSF
Article Snippet: .. Cell culture and flow cytometry Alpha-smooth muscle isoactin positive Human ASMC (Cambrex, Walkersville, MD) in T-75 flasks were starved for 24 hours in 0.1% (vol/vol) fetal bovine serum (FBS) containing medium M199 (Cellgro, Herndon, VA) supplemented with 1% (vol/vol) antibiotic/antimycotic solution (Sigma Chemical Co., St Louis, MO). .. The cells were then stimulated with either vehicle (bovine serum albumin, BSA, vehicle for cytokines (1 mg/ml), in M199; ethanol (EtOH), vehicle for LTD4 (6% final concentration) and PGD2 (0.001–0.01% final concentration); M199, vehicle for tryptase), an individual mediator, or a mediator in combination with GM-CSF at their optimum concentrations for 24 hours.

Cell Culture:

Article Title: Research Upregulation of CD23 (Fc?RII) Expression in Human Airway Smooth Muscle Cells (huASMC) in Response to IL-4, GM-CSF, and IL-4/GM-CSF
Article Snippet: .. Cell culture and flow cytometry Alpha-smooth muscle isoactin positive Human ASMC (Cambrex, Walkersville, MD) in T-75 flasks were starved for 24 hours in 0.1% (vol/vol) fetal bovine serum (FBS) containing medium M199 (Cellgro, Herndon, VA) supplemented with 1% (vol/vol) antibiotic/antimycotic solution (Sigma Chemical Co., St Louis, MO). .. The cells were then stimulated with either vehicle (bovine serum albumin, BSA, vehicle for cytokines (1 mg/ml), in M199; ethanol (EtOH), vehicle for LTD4 (6% final concentration) and PGD2 (0.001–0.01% final concentration); M199, vehicle for tryptase), an individual mediator, or a mediator in combination with GM-CSF at their optimum concentrations for 24 hours.

Article Title: Effect of gold nanoparticle size and coating on labeling monocytes for CT tracking
Article Snippet: .. Cells were cultured with Dubecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (10000 units/MI, 10000 μg/mL), purchased from Cellgro, Corning (Manassas, VA). .. Spherical AuNP of 15 nm and 25 nm in diameter were synthesized using a modified Frens/Turkevich method.

Article Title: Ursolic acid protects monocytes against metabolic stress-induced priming and dysfunction by preventing the induction of Nox4
Article Snippet: .. Briefly, human THP-1 monocytes (ATCC, Manassas, VA) at 1–2×106 cells/ml were cultured at 37 °C for 20 h in RPMI-1640 (Hyclone and Cellgro® ) containing, 10% fetal bovine serum (FBS), 5.5 mM d -glucose, 2% Glutamax, 1% sodium pyruvate (Cellgro® ), 1% penicillin/streptomycin (Cellgro® ), 1% HEPES, 0.1% β-2-mercaptoethanol, and supplemented with either phosphate buffered saline (PBS) or freshly isolated native human LDL (100 µg/ml in PBS) plus d -glucose (high glucose, 20 mM). l -glucose does not increase monocyte priming . .. For selected experiments, peritoneal macrophages were collected from C57BL/6 mice by peritoneal lavage and purified by negative selection using antibody-coated magnetic beads (Dynabeads® mouse pan B (B220) and Dynabeads® mouse pan T (Thy 1.2)).

Article Title: Mycobacterium avium Biofilm Attenuates Mononuclear Phagocyte Function by Triggering Hyperstimulation and Apoptosis during Early Infection
Article Snippet: .. Human THP-1 and natural killer (NK-92) cell lines were obtained from the American Type Culture Collection (Manassas, VA) and always cultured in RPMI 1640 medium supplemented with heat-inactivated 10% fetal bovine serum (FBS), l -glutamine, and 25 mM HEPES (Cellgro, Manassas, VA) incubated at 37°C and in an atmosphere of 5% CO2 . ..

Incubation:

Article Title: Mycobacterium avium Biofilm Attenuates Mononuclear Phagocyte Function by Triggering Hyperstimulation and Apoptosis during Early Infection
Article Snippet: .. Human THP-1 and natural killer (NK-92) cell lines were obtained from the American Type Culture Collection (Manassas, VA) and always cultured in RPMI 1640 medium supplemented with heat-inactivated 10% fetal bovine serum (FBS), l -glutamine, and 25 mM HEPES (Cellgro, Manassas, VA) incubated at 37°C and in an atmosphere of 5% CO2 . ..

other:

Article Title: Differences in gene expression and cytokine production by crystalline vs. amorphous silica in human lung epithelial cells
Article Snippet: Human Bronchial Epithelial Cell Cultures Non-tumorigenic human bronchial epithelial cells (Ad12-SV40 immortalized) BEAS 2B (ATCC, Manassas, VA) were grown and maintained in DMEM/F12 50:50 media containing 10% Fetal Bovine Serum (FBS) (CellGro® Mediatech inc, Manassas, VA), with penicillin (50 units/ml), streptomycin (100 μg/ml) (Invitrogen, Carlsbad, CA), hydrocortisone (100 μg/ml), insulin (2.5 μg/ml), transferrin (2.5 μg/ml) and selenium (2.5 μg/ml) (Sigma, St. Louis, MO).

Modification:

Article Title: Effect of gold nanoparticle size and coating on labeling monocytes for CT tracking
Article Snippet: .. Cells were cultured with Dubecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (10000 units/MI, 10000 μg/mL), purchased from Cellgro, Corning (Manassas, VA). .. Spherical AuNP of 15 nm and 25 nm in diameter were synthesized using a modified Frens/Turkevich method.

Article Title: Grape Seed Proanthocyanidins Induce Apoptosis through p53, Bax, and Caspase 3 Pathways 1
Article Snippet: .. Dulbecco's modified Eagle's medium (DMEM), penicillin, streptomycin, fetal bovine serum (FBS), and trypsin/EDTA were purchased from CellGro (Herndon, VA). .. Acrylamide and the protein assay kit were obtained from Bio-Rad (Hercules, CA).

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • fbs  (Cellgro)
    94
    Cellgro fbs
    Suppression of DEN-2 replication in DCs by siDEN. (A) DCs were isolated from human peripheral blood and cultured in <t>DMEM</t> medium supplemented with <t>FBS,</t> IL-4 and GM-CSF. Non-adherent DCs were harvested on day 7, infected with AAVsiDEN, and two days later the cells were infected with DEN-2 at 0.1 MOI. DCs were harvested 5 days after DEN-2 infection and DEN-2 titers were measured by flow cytometry. (B) Supernatants from DEN-infected DCs were collected and added to culture plates containing confluent Vero cell monolayers. After virus adsorption, the Vero cells were overlaid with agarose and stained with 1% neutral red. Viral plaques were counted 48 h after neutral red overlay. Data are the averages of two independent experiments. * p
    Fbs, supplied by Cellgro, used in various techniques. Bioz Stars score: 94/100, based on 162 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fbs/product/Cellgro
    Average 94 stars, based on 162 article reviews
    Price from $9.99 to $1999.99
    fbs - by Bioz Stars, 2020-07
    94/100 stars
      Buy from Supplier

    92
    Cellgro heat inactivated fetal bovine serum fbs
    S1P from platelets after PDPN-CLEC-2-dependent activation contributes to HEV barrier function a, ELISAs of S1P concentrations in supernatants of WT and Clec-2 − / − platelets after incubation with CLEC-2 activating antibody, INU1 (INU1-treated) or isotype control rat IgG (sham-treated). S1P in platelet lysates (plt lysates) was used as the positive control (mean ± s.d., n = 4 mice per group representing two individual experiments). b, Representative images of VE-cad staining of HEVs from WT LN slices incubated for 1.5 hrs with <t>DMEM</t> and normal <t>FBS</t> (media), DMEM and lipid-depleted FBS (lipid-depleted), lipid-depleted and WT platelets (+WT plts), lipid-depleted and Clec-2 − / − platelets (+ Clec-2 − / − plts), lipid-depleted and S1Pless platelets (+S1Pless plts), or lipid-depleted and WT plts plus S1PR1 antagonist W146 (+WT plts/W146). Pdpn f/f ;CagCre LN slices incubated with lipid-depleted and WT platelets (+WT plts) were controls. 100 HEVs analyzed per condition. Dashed lines mark HEVs. # marks non-specific staining as also observed in negative controls. Graphs represent ratios of VE-cad intensities on HEVs relative to that of WT LN slices cultured with media (mean ± s.d., n = 20 HEVs/group). c, Gross morphology (insets) and confocal images of draining popliteal LNs (PLN) after immunisation. Asterisk indicates bleeding. LV, lymphatic vessel (Lyve-1 + ). Arrow marks bleeding (Ter119 + ) around an HEV (CD31 + ). d , Confocal images of PLN HEVs from S1Pless mice transfused with Clec-2 − / − or WT platelets (plts) for 4 days and littermate controls transfused with WT plts after intravenous FITC-dextran injection. Arrows indicate vascular leak of FITC-dextran. Graphs on right quantify leaking HEVs (mean ± s.d., 50 HEVs/group ( n = 3)). e , Model depicting how PDPN maintains HEV integrity during lymphocyte trafficking. FRC PDPN engages CLEC-2 on extravasated platelets in the perivenular space of HEVs and induces local release of S1P, which promotes VE-cad expression on the WT HEV (left). In contrast, loss of the interaction results in impaired HEV integrity and subsequent bleeding (right). b–d, Data is from three individual experiments. Scale bars, 50 μm. ***, P
    Heat Inactivated Fetal Bovine Serum Fbs, supplied by Cellgro, used in various techniques. Bioz Stars score: 92/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/heat inactivated fetal bovine serum fbs/product/Cellgro
    Average 92 stars, based on 23 article reviews
    Price from $9.99 to $1999.99
    heat inactivated fetal bovine serum fbs - by Bioz Stars, 2020-07
    92/100 stars
      Buy from Supplier

    Image Search Results


    Suppression of DEN-2 replication in DCs by siDEN. (A) DCs were isolated from human peripheral blood and cultured in DMEM medium supplemented with FBS, IL-4 and GM-CSF. Non-adherent DCs were harvested on day 7, infected with AAVsiDEN, and two days later the cells were infected with DEN-2 at 0.1 MOI. DCs were harvested 5 days after DEN-2 infection and DEN-2 titers were measured by flow cytometry. (B) Supernatants from DEN-infected DCs were collected and added to culture plates containing confluent Vero cell monolayers. After virus adsorption, the Vero cells were overlaid with agarose and stained with 1% neutral red. Viral plaques were counted 48 h after neutral red overlay. Data are the averages of two independent experiments. * p

    Journal: Genetic Vaccines and Therapy

    Article Title: Attenuation of dengue virus infection by adeno-associated virus-mediated siRNA delivery

    doi: 10.1186/1479-0556-2-8

    Figure Lengend Snippet: Suppression of DEN-2 replication in DCs by siDEN. (A) DCs were isolated from human peripheral blood and cultured in DMEM medium supplemented with FBS, IL-4 and GM-CSF. Non-adherent DCs were harvested on day 7, infected with AAVsiDEN, and two days later the cells were infected with DEN-2 at 0.1 MOI. DCs were harvested 5 days after DEN-2 infection and DEN-2 titers were measured by flow cytometry. (B) Supernatants from DEN-infected DCs were collected and added to culture plates containing confluent Vero cell monolayers. After virus adsorption, the Vero cells were overlaid with agarose and stained with 1% neutral red. Viral plaques were counted 48 h after neutral red overlay. Data are the averages of two independent experiments. * p

    Article Snippet: After 2 h at 37°C/5%CO2 the nonadherent cells were removed and the adherent cells were cultured with fresh DMEM supplemented with 10% FBS (Cellgro), 200 ng/ml IL-4 (BD-Pharmingen) and 50 ng/ml GM-CSF (BD-Pharmingen) for 7 days prior to infection with DEN.

    Techniques: Isolation, Cell Culture, Infection, Flow Cytometry, Cytometry, Adsorption, Staining

    GSP induces significantly less apoptosis in p53-deficient (p53 -/- ) cells compared to p53 wild-type (p53 +/+ ) cells. Both p53 +/+ and p53 -/- fibroblast cells were cultured under identical conditions as was done in JB6 C141 cells except that the cells were treated with 15% FBS in DMEM. Cells were starved in 0.5% FBS/DMEM overnight and thereafter treated with GSP in serum-containing media for another 24 hours. Morphologic changes in p53 +/+ and p53 -/- fibroblast cells undergoing apoptosis were observed under fluorescence microscopy. Cells in panels A and G were not treated with GSP (controls), whereas increasing concentrations of GSP were added to cultures for 24 hours after overnight serum starvation in panels B–E and H–K by 20, 40, 60, and 80 µg/ml, respectively. The percentage of apoptotic cells from panel A to panel E is summarized in panel F, and the percentage of apoptotic cells from panel G to panel K is summarized in panel L. The experiment was repeated at least two times and the average percentage of apoptotic cells ± SD for each treatment group is indicated. At least 200 cells were counted in a blinded manner to score the percentage of apoptosis in each treatment group. *P

    Journal: Neoplasia (New York, N.Y.)

    Article Title: Grape Seed Proanthocyanidins Induce Apoptosis through p53, Bax, and Caspase 3 Pathways 1

    doi:

    Figure Lengend Snippet: GSP induces significantly less apoptosis in p53-deficient (p53 -/- ) cells compared to p53 wild-type (p53 +/+ ) cells. Both p53 +/+ and p53 -/- fibroblast cells were cultured under identical conditions as was done in JB6 C141 cells except that the cells were treated with 15% FBS in DMEM. Cells were starved in 0.5% FBS/DMEM overnight and thereafter treated with GSP in serum-containing media for another 24 hours. Morphologic changes in p53 +/+ and p53 -/- fibroblast cells undergoing apoptosis were observed under fluorescence microscopy. Cells in panels A and G were not treated with GSP (controls), whereas increasing concentrations of GSP were added to cultures for 24 hours after overnight serum starvation in panels B–E and H–K by 20, 40, 60, and 80 µg/ml, respectively. The percentage of apoptotic cells from panel A to panel E is summarized in panel F, and the percentage of apoptotic cells from panel G to panel K is summarized in panel L. The experiment was repeated at least two times and the average percentage of apoptotic cells ± SD for each treatment group is indicated. At least 200 cells were counted in a blinded manner to score the percentage of apoptosis in each treatment group. *P

    Article Snippet: Dulbecco's modified Eagle's medium (DMEM), penicillin, streptomycin, fetal bovine serum (FBS), and trypsin/EDTA were purchased from CellGro (Herndon, VA).

    Techniques: Cell Culture, Fluorescence, Microscopy

    Treatment of GSP decreases the expression of the antiapoptotic proteins, Bcl-2 and Bcl-xl, and increases the expression of the pro-apoptotic protein, Bax, in JB6 C141 cells. JB6 C141 cells were starved in 0.5% FBS/DMEM overnight and then treated with GSP (20–80 µg/ml) in serum-containing media for another 24 hours. Cell lysates were prepared, and the expression of the proteins was determined by Western blot analysis using the corresponding antibodies, as detailed in the Materials and Methods section. Relative intensity of bands in each panel was determined. GSP treatment to JB6 C141 cells decreased the expressions of the antiapoptotic proteins, Bcl-2 (panel A) and Bcl-xl (panel D), whereas the expression of the pro-apoptotic protein, Bax, was increased (panel B). A representative blot is shown from three independent experiments with identical results. The ratio of Bax and Bcl-2 protein expression was determined from three separate experiments by comparing the relative intensities of protein bands and shown as a mean ± SD (panel C). β-Actin was used as an internal control to monitor equal protein loading and transfer of proteins from the gel to the membranes after stripping them and reprobing them with the actin antibody.

    Journal: Neoplasia (New York, N.Y.)

    Article Title: Grape Seed Proanthocyanidins Induce Apoptosis through p53, Bax, and Caspase 3 Pathways 1

    doi:

    Figure Lengend Snippet: Treatment of GSP decreases the expression of the antiapoptotic proteins, Bcl-2 and Bcl-xl, and increases the expression of the pro-apoptotic protein, Bax, in JB6 C141 cells. JB6 C141 cells were starved in 0.5% FBS/DMEM overnight and then treated with GSP (20–80 µg/ml) in serum-containing media for another 24 hours. Cell lysates were prepared, and the expression of the proteins was determined by Western blot analysis using the corresponding antibodies, as detailed in the Materials and Methods section. Relative intensity of bands in each panel was determined. GSP treatment to JB6 C141 cells decreased the expressions of the antiapoptotic proteins, Bcl-2 (panel A) and Bcl-xl (panel D), whereas the expression of the pro-apoptotic protein, Bax, was increased (panel B). A representative blot is shown from three independent experiments with identical results. The ratio of Bax and Bcl-2 protein expression was determined from three separate experiments by comparing the relative intensities of protein bands and shown as a mean ± SD (panel C). β-Actin was used as an internal control to monitor equal protein loading and transfer of proteins from the gel to the membranes after stripping them and reprobing them with the actin antibody.

    Article Snippet: Dulbecco's modified Eagle's medium (DMEM), penicillin, streptomycin, fetal bovine serum (FBS), and trypsin/EDTA were purchased from CellGro (Herndon, VA).

    Techniques: Expressing, Western Blot, Stripping Membranes

    S1P from platelets after PDPN-CLEC-2-dependent activation contributes to HEV barrier function a, ELISAs of S1P concentrations in supernatants of WT and Clec-2 − / − platelets after incubation with CLEC-2 activating antibody, INU1 (INU1-treated) or isotype control rat IgG (sham-treated). S1P in platelet lysates (plt lysates) was used as the positive control (mean ± s.d., n = 4 mice per group representing two individual experiments). b, Representative images of VE-cad staining of HEVs from WT LN slices incubated for 1.5 hrs with DMEM and normal FBS (media), DMEM and lipid-depleted FBS (lipid-depleted), lipid-depleted and WT platelets (+WT plts), lipid-depleted and Clec-2 − / − platelets (+ Clec-2 − / − plts), lipid-depleted and S1Pless platelets (+S1Pless plts), or lipid-depleted and WT plts plus S1PR1 antagonist W146 (+WT plts/W146). Pdpn f/f ;CagCre LN slices incubated with lipid-depleted and WT platelets (+WT plts) were controls. 100 HEVs analyzed per condition. Dashed lines mark HEVs. # marks non-specific staining as also observed in negative controls. Graphs represent ratios of VE-cad intensities on HEVs relative to that of WT LN slices cultured with media (mean ± s.d., n = 20 HEVs/group). c, Gross morphology (insets) and confocal images of draining popliteal LNs (PLN) after immunisation. Asterisk indicates bleeding. LV, lymphatic vessel (Lyve-1 + ). Arrow marks bleeding (Ter119 + ) around an HEV (CD31 + ). d , Confocal images of PLN HEVs from S1Pless mice transfused with Clec-2 − / − or WT platelets (plts) for 4 days and littermate controls transfused with WT plts after intravenous FITC-dextran injection. Arrows indicate vascular leak of FITC-dextran. Graphs on right quantify leaking HEVs (mean ± s.d., 50 HEVs/group ( n = 3)). e , Model depicting how PDPN maintains HEV integrity during lymphocyte trafficking. FRC PDPN engages CLEC-2 on extravasated platelets in the perivenular space of HEVs and induces local release of S1P, which promotes VE-cad expression on the WT HEV (left). In contrast, loss of the interaction results in impaired HEV integrity and subsequent bleeding (right). b–d, Data is from three individual experiments. Scale bars, 50 μm. ***, P

    Journal: Nature

    Article Title: Podoplanin maintains high endothelial venule integrity by interacting with platelet CLEC-2

    doi: 10.1038/nature12501

    Figure Lengend Snippet: S1P from platelets after PDPN-CLEC-2-dependent activation contributes to HEV barrier function a, ELISAs of S1P concentrations in supernatants of WT and Clec-2 − / − platelets after incubation with CLEC-2 activating antibody, INU1 (INU1-treated) or isotype control rat IgG (sham-treated). S1P in platelet lysates (plt lysates) was used as the positive control (mean ± s.d., n = 4 mice per group representing two individual experiments). b, Representative images of VE-cad staining of HEVs from WT LN slices incubated for 1.5 hrs with DMEM and normal FBS (media), DMEM and lipid-depleted FBS (lipid-depleted), lipid-depleted and WT platelets (+WT plts), lipid-depleted and Clec-2 − / − platelets (+ Clec-2 − / − plts), lipid-depleted and S1Pless platelets (+S1Pless plts), or lipid-depleted and WT plts plus S1PR1 antagonist W146 (+WT plts/W146). Pdpn f/f ;CagCre LN slices incubated with lipid-depleted and WT platelets (+WT plts) were controls. 100 HEVs analyzed per condition. Dashed lines mark HEVs. # marks non-specific staining as also observed in negative controls. Graphs represent ratios of VE-cad intensities on HEVs relative to that of WT LN slices cultured with media (mean ± s.d., n = 20 HEVs/group). c, Gross morphology (insets) and confocal images of draining popliteal LNs (PLN) after immunisation. Asterisk indicates bleeding. LV, lymphatic vessel (Lyve-1 + ). Arrow marks bleeding (Ter119 + ) around an HEV (CD31 + ). d , Confocal images of PLN HEVs from S1Pless mice transfused with Clec-2 − / − or WT platelets (plts) for 4 days and littermate controls transfused with WT plts after intravenous FITC-dextran injection. Arrows indicate vascular leak of FITC-dextran. Graphs on right quantify leaking HEVs (mean ± s.d., 50 HEVs/group ( n = 3)). e , Model depicting how PDPN maintains HEV integrity during lymphocyte trafficking. FRC PDPN engages CLEC-2 on extravasated platelets in the perivenular space of HEVs and induces local release of S1P, which promotes VE-cad expression on the WT HEV (left). In contrast, loss of the interaction results in impaired HEV integrity and subsequent bleeding (right). b–d, Data is from three individual experiments. Scale bars, 50 μm. ***, P

    Article Snippet: Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% heat-inactivated fetal bovine serum (FBS) and 1% L-glutamine/penicillin/streptomycin (Cellgro) at 37°C in a humidified atmosphere of 5% CO2 .

    Techniques: Activation Assay, Incubation, Positive Control, Mouse Assay, Staining, Cell Culture, Injection, Expressing