rbcs  (MedChemExpress)

Journal: Lab on a Chip

Article Title: Evaluating anti-sickling therapies for sickle cell disease: a microfluidic assay for red blood cell-mediated microvascular occlusion under hypoxia

doi: 10.1039/d5lc00264h

Figure Lengend Snippet: Schematic illustration of occlusion devices used to assess hypoxia-induced red RBC sickling. (A) Assembly of the OcclusionChip, featuring microcapillaries with diameters decreasing from 20 μm to 4 μm along the flow direction to retain deformed RBCs. (B) 4 μm micropillar array demonstrating that non-deformed HbSS RBCs traverse the micropillars under normoxic conditions, whereas deformed HbSS RBCs occlude the micropillars under induced hypoxia. (C) Sequential chemical reaction of SMB in PBS-G buffer eliminates extracellular oxygen (O 2 ), creating a hypoxic environment necessary for RBC sickling. (D) Experimental setup for chemically inducing hypoxia-mediated sickling: RBC suspension was combined with SMB buffer and incubated for 5 minutes to achieve depletion in partial pressure of oxygen of p O 2 ∼ 44 mmHg. Deoxygenated RBCs were perfused into the OcclusionChip using a constant inlet pressure pump set at 50 mbar for 10 minutes. Images of the OcclusionChip mounted on an inverted microscope were captured, and the percentage of OI was quantified using cell counting software.

Article Snippet: For RBCs treated with PKR-3 (HbSS + PKR-3) or etavopivat (HbSS + FT-4202), packed RBCs at 20% hematocrit in PBS-G were incubated for 6 hours at 37 °C with 200 μM PKR-3 (MedChemExpress, PKR-IN-2, Cat No. HY-19702) or FT-4202 (MedChemExpress, FT-4202, Cat No. HY-139573) dissolved in DMSO.

Techniques: Suspension, Incubation, Inverted Microscopy, Cell Counting, Software

Journal: Lab on a Chip

Article Title: Evaluating anti-sickling therapies for sickle cell disease: a microfluidic assay for red blood cell-mediated microvascular occlusion under hypoxia

doi: 10.1039/d5lc00264h

Figure Lengend Snippet: Optimization of chemically induced hypoxia-mediated occlusion in the OcclusionChip using sodium metabisulphite (SMB) compared to nitrogen gas-induced hypoxia. (A) Reducing SMB concentration from 5% to 1% and incubation time from 50 to 5 minutes at 20% hematocrit achieved optimal HOI conditions comparable to nitrogen-induced hypoxia. (B) For HbSS RBCs, HOI at 1.5% SMB and 5-minute incubation was not significantly different from nitrogen-induced HOI (8% difference; p = 0.58, n = 4, Wilcoxon test). However, HOI at 1% SMB was significantly lower, showing a 25% difference compared to nitrogen-induced HOI ( p = 0.007, n = 4, Wilcoxon test). (C) For HbSS RBCs, at 1.5% SMS, HOI values showed no significant differences between 10, 12, and 15 minutes of RBC perfusion into the OcclusionChip ( p > 0.05 for all comparisons, n = 5, Friedman-test). (D) p O 2 kinetics under chemically induced hypoxia: incubation of HbAA RBCs with 1.5% SMB for 5 min lowered p O 2 from 100 mmHg to ∼44 mmHg; perfusion into the OcclusionChip further decreased p O 2 from 44 mmHg to ∼35 mmHg by 10 min, where it stabilized between 32–23 mmHg through 20 min ( n = 5). The blue line represents measured p O 2 data for a representative HbAA sample. (E) Representative microcapillary occlusion images from HbSS and HbAA subjects under normoxia, nitrogen-induced hypoxia, and chemically induced hypoxia. (F) Comparison of microcapillary HOI showed no significant differences for HbAA RBCs between normoxic and hypoxic conditions ( p = 0.50, n = 6, Wilcoxon test). HbSS RBCs exhibited significantly increased HOI under both nitrogen-induced and chemically induced hypoxia compared to normoxia ( p = 0.007, n = 15, paired t -test). There were no significant differences between HOI values under nitrogen-induced hypoxia and chemically induced hypoxia for both HbAA ( p = 0.90, n = 6, paired t -tests) and HbSS RBCs ( p = 0.98; n = 15, paired t -tests). HbSS RBCs had significantly higher HOI compared to HbAA RBCs under chemically induced hypoxia ( p = 0.001; HbAA n = 5, HbSS n = 15; Mann–Whitney test). n represents RBCs from different individuals. Error bars are presented as SD.

Article Snippet: For RBCs treated with PKR-3 (HbSS + PKR-3) or etavopivat (HbSS + FT-4202), packed RBCs at 20% hematocrit in PBS-G were incubated for 6 hours at 37 °C with 200 μM PKR-3 (MedChemExpress, PKR-IN-2, Cat No. HY-19702) or FT-4202 (MedChemExpress, FT-4202, Cat No. HY-139573) dissolved in DMSO.

Techniques: Concentration Assay, Incubation, Comparison, MANN-WHITNEY

Journal: Lab on a Chip

Article Title: Evaluating anti-sickling therapies for sickle cell disease: a microfluidic assay for red blood cell-mediated microvascular occlusion under hypoxia

doi: 10.1039/d5lc00264h

Figure Lengend Snippet: HOI detects hemoglobin S (HbS)-related RBC abnormalities and correlates with clinical parameters in subjects with HbSS and HbSC. (A) HOI significantly increased in HbS carriers: (HbAS: n = 15, p = 0.003; HbSC: n = 5, p = 0.002; HbSS: n = 14, p = 0.001; paired t -test), whereas HbAA RBCs showed no significant response ( n = 5, p = 0.18, paired t -test). (B) HOI positively correlated with HbS levels in HbS-carrying RBCs: HbSS ( n = 14), HbAS ( n = 15), and HbSC ( n = 5) ( R 2 = 0.65, p = 0.001). In subjects with HbSS ( n = 14) and HbSC ( n = 5), HOI was significantly associated with: (C) lactate dehydrogenase (LDH) levels ( R 2 = 0.67, p = 0.001), (D) white blood cell (WBC) count ( R 2 = 0.73, p = 0.006), (E) reticulocyte count ( R 2 = 0.81, p = 0.001), and (F) platelet count ( R 2 = 0.33, p = 0.004). Each symbol represents a different genotype. n represents RBCs from different individuals. Error bars are presented as mean ± SD.

Article Snippet: For RBCs treated with PKR-3 (HbSS + PKR-3) or etavopivat (HbSS + FT-4202), packed RBCs at 20% hematocrit in PBS-G were incubated for 6 hours at 37 °C with 200 μM PKR-3 (MedChemExpress, PKR-IN-2, Cat No. HY-19702) or FT-4202 (MedChemExpress, FT-4202, Cat No. HY-139573) dissolved in DMSO.

Techniques:

Journal: Lab on a Chip

Article Title: Evaluating anti-sickling therapies for sickle cell disease: a microfluidic assay for red blood cell-mediated microvascular occlusion under hypoxia

doi: 10.1039/d5lc00264h

Figure Lengend Snippet: HOI used as a tool for evaluating therapeutic interventions in hemoglobin S (HbS) – carrying RBCs. (A) HbSS RBCs ( n = 9, each patient sample is represented by an empty symbol): Treatment with the oxygen modifiers GBT021601, GBT440, and the PKR activators PKR-3 and FT4202 each significantly reduced HOI compared to untreated cells. The GBT021601 + FT4202 combination further decreased HOI versus untreated RBCs and versus FT4202 alone but did not differ from GBT021601 alone. (B) HbSC RBCs ( n = 9, each patient sample is represented by a filled symbol): GBT021601, GBT440, PKR-3, and FT4202 each lowered HOI relative to untreated controls. The GBT021601 + FT4202 combination further reduced HOI versus untreated RBCs and versus GBT021601 alone, with no significant difference from FT4202 alone. (C) HbSS RBCs from patients on transfusion therapy (TX) or hydroxyurea (HU) ( n = 7; each patient sample is represented by an empty symbol): ex vivo addition of GBT440 or PKR-3 significantly lowered HOI compared to TX or HU-treated samples alone. For (A–C), statistics were performed using the paired test and the Wilcoxon test *** p < 0.001, ** p < 0.01, * p < 0.05, ns not statistically significant. n represents RBCs from different individuals. Data are shown as mean ± SD.

Article Snippet: For RBCs treated with PKR-3 (HbSS + PKR-3) or etavopivat (HbSS + FT-4202), packed RBCs at 20% hematocrit in PBS-G were incubated for 6 hours at 37 °C with 200 μM PKR-3 (MedChemExpress, PKR-IN-2, Cat No. HY-19702) or FT-4202 (MedChemExpress, FT-4202, Cat No. HY-139573) dissolved in DMSO.

Techniques: Ex Vivo

Journal: Molecular Vision

Article Title: PKR promotes choroidal neovascularization via upregulating the PI3K/Akt signaling pathway in VEGF expression

doi:

Figure Lengend Snippet: Effect of PKR siRNA and PI3K inhibitor LY294002 on hypoxia-induced PKR, p-PI3K, p-Akt, and VEGF expression in RF/6A cells. A : DsRNA-activated protein kinase (PKR), phosphophosphatidylinositol 3-kinase (p-PI3K), phosphoprotein kinase B (p-Akt), and vascular endothelial growth factor (VEGF) expression was detected with western blotting in the RF/6A cells following PKR siRNA transfection after 48 h. C : The histogram shows the densitometric analysis of the average levels for PKR, p-PI3K, p-Akt, and VEGF to GAPDH. Cells transfected with scramble siRNA were used as the negative control. B : The western blot shows PKR, p-PI3K, p-Akt, and VEGF expression in RF/6A cells treated with the PI3K inhibitor LY294002 for 30 min. D : The histogram shows the densitometric analysis of the average levels of PKR, p-PI3K, p-Akt, and VEGF to GAPDH. Hypoxic cells were used as the negative control. *p<0.05, statistically significantly different compared to the respective controls. Values represent means ± SD.

Article Snippet: Transfection of PKR siRNA (5′-GGA TTC GGG TTA CTT GTA A-3′, RiboBio, Guangzhou, China) was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) following the manufacturer’s protocol.

Techniques: Expressing, Western Blot, Transfection, Negative Control

Journal: Molecular Vision

Article Title: PKR promotes choroidal neovascularization via upregulating the PI3K/Akt signaling pathway in VEGF expression

doi:

Figure Lengend Snippet: PKR promotes migration of RF/6A cells in a coculture system under hypoxic conditions. A : Crystal violet staining detected the migrated cells of each group: normal, hypoxic, scramble siRNA, and dsRNA-activated protein kinase (PKR) siRNA. Representative photographs of migrated RF/6A cells (200× magnification). B : The average number of migrated RF/6A cells per field. *p<0.01, PKR siRNA group versus the hypoxic group. Values represent means ± SD.

Article Snippet: Transfection of PKR siRNA (5′-GGA TTC GGG TTA CTT GTA A-3′, RiboBio, Guangzhou, China) was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) following the manufacturer’s protocol.

Techniques: Migration, Staining

Journal: Molecular Vision

Article Title: PKR promotes choroidal neovascularization via upregulating the PI3K/Akt signaling pathway in VEGF expression

doi:

Figure Lengend Snippet: PKR promotes tube formation of RF/6A cells in a coculture system under hypoxic conditions. A : Microscopic images showing tube formation in each group: normal, hypoxic, scramble siRNA, and dsRNA-activated protein kinase (PKR) siRNA. Representative photographs of tube formation of RF/6A cells (200× magnification). B : Decreased tube formation was observed when the cocultured RF/6A cells were transfected with PKR siRNA under hypoxia. *p<0.01, PKR siRNA group versus the hypoxia group. Values represent means ± SD.

Article Snippet: Transfection of PKR siRNA (5′-GGA TTC GGG TTA CTT GTA A-3′, RiboBio, Guangzhou, China) was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) following the manufacturer’s protocol.

Techniques: Transfection