Journal: BMC Veterinary Research

Article Title: Neural stem/progenitor cells from adult canine cervical spinal cord have the potential to differentiate into neural lineage cells

doi: 10.1186/s12917-023-03757-3

Figure Lengend Snippet: Immunocytochemistry of neural stem/progenitor cells (NSPCs) cultured as neurospheres or monolayers. (A) 3rd, 6th, and 9th passage cells cultured in a monolayer for 5 DIV. (B) 3rd, 6th, and 9th passage cells cultured as neurosphere for 5 DIV. Nuclei were stained with DAPI (blue) and NSPCs marker for SOX2 (green) and nestin (red). (C) Each well of the 24-well plate was immunostained with nestin and SOX2. The quantification was performed by calculating the average percentage of nestin + /SOX2 + cells out of the total cells in three wells. Quantification was carried out with cells cultured in monolayer

Article Snippet: The following primary antibodies were used: nestin (1:400; sc-33,677; Santa Cruz Biotechnology, Dallas, TX, USA) and SOX2 (1:400; bs-23176R-A488; Bioss, Massachusetts, USA) (neural progenitor markers); GFAP (1:400; sc-33,673; Santa Cruz Biotechnology) (glial marker); MBP (1:400; bs-0380R-A488; Bioss) and β3-tubulin (1:400; sc-80,005; Santa Cruz Biotechnology) (neuronal markers).

Techniques: Immunocytochemistry, Cell Culture, Staining, Marker

Journal: BMC Veterinary Research

Article Title: Neural stem/progenitor cells from adult canine cervical spinal cord have the potential to differentiate into neural lineage cells

doi: 10.1186/s12917-023-03757-3

Figure Lengend Snippet: Primer sequences used for real-time quantitative PCR

Article Snippet: The following primary antibodies were used: nestin (1:400; sc-33,677; Santa Cruz Biotechnology, Dallas, TX, USA) and SOX2 (1:400; bs-23176R-A488; Bioss, Massachusetts, USA) (neural progenitor markers); GFAP (1:400; sc-33,673; Santa Cruz Biotechnology) (glial marker); MBP (1:400; bs-0380R-A488; Bioss) and β3-tubulin (1:400; sc-80,005; Santa Cruz Biotechnology) (neuronal markers).

Techniques: Sequencing

Journal: Nanomaterials

Article Title: Inhibition of TRPV1 Channel Activity in Human CD4 + T Cells by Nanodiamond and Nanoplatinum Liquid, DPV576

doi: 10.3390/nano8100770

Figure Lengend Snippet: ( A – D ) DPV576 does not modulate the expression of TRPV4 on CD4 + T cells. Inactivated and anti-CD3/CD28 activated CD4 + T lymphocytes were exposed to DPV576 for 24 h. The cells were stained for TRPV channels. Expression of TRPV4 on non-activated T cells ( A , B ). Anti-CD3/CD28 activated T cells ( C , D ). Data is representative of three experiments. Blue: Without DPV576; Red: DPV576 exposed lymphocytes; Green: Isotype.

Article Snippet: Briefly, cells were collected and centrifuged, re-suspended in PBS (phosphate buffered saline) 2% FBS, and incubated with specific antibodies for TRPV1-AL647 (bs-1931R-A647) or TRPV4-Alexa 488 (bs-6425R-A488) (Bioss Inc., Woburn, MA, USA) at a dilution of 1:100 for 1 h. Subsequently, the cells were washed and a minimum of 10,000 cells were acquired on FACS Calibur (Becton Dickinson, San Jose, CA, USA).

Techniques: Expressing, Staining

Journal: Clinical & Translational Immunology

Article Title: Blunted sFasL signalling exacerbates TNF‐driven neutrophil necroptosis in critically ill COVID‐19 patients

doi: 10.1002/cti2.1357

Figure Lengend Snippet: Neutrophil necroptosis through the RIPK1‐RIPK3‐MLKL axis in COVID‐19. (a–d) Representative flow cytometry plots at 4 h (a) or 18 h post‐stimulation, (c) and quantification of live COVID‐19 ( n = 7 or 8) or healthy donor ( n = 7) neutrophils stimulated with autologous or heterologous plasma, untreated or treated with 50 µM Q‐VD‐Oph for 4 h (b) or 18 h (d) . (e–g) Analysis of LDH and DNA, (e) and DAMPs (f) and cytokines (g) in supernatants of COVID‐19 ( n = 7 or 13) or healthy donor ( n = 6 or 7) neutrophils stimulated with autologous or heterologous plasma for 4 h. (h, i) Representative histogram and quantification of GMFI of intracellular RIPK1 expression (h) and caspase‐8 activity (i) of COVID‐19 ( n = 9) or healthy donor ( n = 6 or 7) neutrophils stimulated with autologous or heterologous plasma for 4 h. ( j ) Quantification of live (Annexin V − /7AAD − ), dying (Annexin V + /7AAD − ) and dead (Annexin V + /7AAD + ) healthy donor neutrophils ( n = 4) stimulated with healthy or COVID‐19 plasma ( n = 12), untreated or treated with 5 µM NSA for 4 h. For each experiment, healthy donor neutrophils were stimulated separately with plasma from three different COVID‐19 patients. Each square or connected squares represent one donor. Shown are mean ± SEM. Statistics were calculated by the paired t ‐test or the Wilcoxon signed‐rank test. P ‐values are indicated within the graphs. (k) RIPK1, RIPK3, MLKL and pMLKL staining of COVID‐19 ( n = 2) and non‐COVID‐19 thrombi. Scale bar, 50 μm. Arrows indicate strong positive staining, and arrowheads indicate negative or weak positive staining. NSA, necrosulphonamide; SEM, standard error of means.

Article Snippet: Next, staining with anti‐FasL BV421 (NOK‐1) from Biolegend or anti‐RIPK1 AF488 (Polyclonal) from Bioss Antibodies (Woburn MA, USA) was carried out in Fix/Perm solution B for 30 min at 4°C.

Techniques: Flow Cytometry, Expressing, Activity Assay, Staining

Journal: Clinical & Translational Immunology

Article Title: Blunted sFasL signalling exacerbates TNF‐driven neutrophil necroptosis in critically ill COVID‐19 patients

doi: 10.1002/cti2.1357

Figure Lengend Snippet: Impaired sFasL signalling during COVID‐19 favors RIPK1‐driven necroptosis. (a) TNFRI expression on COVID‐19 ( n = 9) and healthy donor neutrophils ( n = 8). (b, c) Quantification of live (Annexin V − /7AAD − ), dying (Annexin V + /7AAD − ) and dead (Annexin V + /7AAD + ) healthy donor neutrophils ( n = 4) stimulated with healthy or COVID‐19 plasma ( n = 12), untreated or treated with 2 µg mL −1 anti‐TNFRI (b) or with 30 ng mL −1 TNF‐α with or without 5 µM NSA (c) . For each experiment, healthy donor neutrophils were stimulated separately with plasma from three different COVID‐19 patients. (d) Luminex‐based analysis of COVID‐19 (sFasL, n = 56/61; TRAIL, n = 28/61 detected) and healthy donors' plasma (sFasL, n = 22/22; TRAIL, n = 17/22). (e) Representative histogram and quantification of receptor expression on neutrophils from COVID‐19 ( n = 9) and healthy donors ( n = 8). (f, g) Luminex‐based analysis of sFasL in supernatants (f) and quantification of intracellular FasL expression (g) of COVID‐19 neutrophils ( n = 8 or 9) stimulated with autologous or heterologous plasma for 4 h. (h, i) Representative flow cytometry plots (h) and quantification of live, dying and dead (i) COVID‐19 neutrophils ( n = 9) stimulated with autologous or heterologous plasma and with or without 100 ng mL ‐1 sFasL for 4 h. (j, k) Representative flow cytometry plots (j) and quantification of live, dying and dead (k) COVID‐19 neutrophils ( n = 9) stimulated with autologous or heterologous plasma and with or without 10 μg mL −1 anti‐Fas for 4 h. (l–n) Representative histogram and quantification of GMFI of intracellular RIPK1 expression (l) , caspase‐8 activity (m) or caspase‐3/7 activity (n) of COVID‐19 neutrophils ( n = 8 or 9) stimulated with autologous or heterologous plasma and with or without 100 ng mL −1 sFasL for 4 h. Each dot, each square or connected squares represent one donor. Shown are mean ± SEM. Statistics were calculated by the unpaired t ‐test, Mann–Whitney U ‐test, paired t ‐test or Wilcoxon signed‐rank test. P ‐values are indicated within the graphs. NSA, necrosulphonamide; SEM, standard error of means.

Article Snippet: Next, staining with anti‐FasL BV421 (NOK‐1) from Biolegend or anti‐RIPK1 AF488 (Polyclonal) from Bioss Antibodies (Woburn MA, USA) was carried out in Fix/Perm solution B for 30 min at 4°C.

Techniques: Expressing, Luminex, Flow Cytometry, Activity Assay, MANN-WHITNEY

Journal: International Journal of Nanomedicine

Article Title: Long Non-Coding RNAs Within Macrophage-Derived Exosomes Promote BMSC Osteogenesis in a Bone Fracture Rat Model

doi: 10.2147/IJN.S398446

Figure Lengend Snippet: The characteristics of macrophages and BMSCs. ( a ) Primary macrophages observed under the microscope; cells were spherical and uniform in size. ( b ) Flow cytometry results of CD68 expression on the cell surface. The expression of CD68 on 10,000 cells was recorded. 90.7% of the cells expressed CD68. ( c ) The fifth generation of cultured BMSCs was observed under the microscope; the cells were long, fusiform, and translucent. ( d – f ) Alizarin red, Alcian blue, and Oil red O staining showed that the cells had osteogenic, chondrogenic, and adipogenic differentiation ability. ( g – j ). The expression of CD29, CD90, CD44, and CD34 on 10,000 cells was recorded; red indicates cell marker expression and blue indicates the isotype control. Microscopy: 100× magnification and 200 μm scale.

Article Snippet: Cells were incubated with the following specific antibodies in the dark at 4 °C for 30 min: rabbit anti-CD29/Alexa Fluor 488 (1:100 dilution), rabbit anti-CD90/Alexa Fluor 488 (1:100 dilution), rabbit anti-CD44/Alexa Fluor 488 (1:100 dilution), rabbit anti-CD34/Alexa Fluor 488 (1:100 dilution), and rabbit anti-CD68/Alexa Fluor 488 (1:100 dilution) (all purchased from Bioss, China).

Techniques: Microscopy, Flow Cytometry, Expressing, Cell Culture, Staining, Marker