The manipulated T-ALL cells were pre-stained with PKH26 followed by the treatment with PBS, hCD28-mAb (R&D Systems, 0.5 μg/mL), rhCD40 (Abcam, 0.5 μg/mL) or both (hCD28-mAb+rhCD40) in RPMI1640 with no FBS. Cells were then added to transwell inserts with 8 μm pore size polycarbonate filters (Corning, Kennebunk, ME, USA) in a 24-well plate. Culture media containing the pre-established monolayer of HS-5 BMSCs or complete media with recombinant human CXCL12 (rhCXCL12, R&D Systems, 10 ng/mL) were placed in the lower wells. Chambers were incubated for 4 hours at 37 °C in 5% CO2, and cell migration was quantified by measuring PKH26 dye intensity in lower chambers.
Rhcxcl12
Manufactured by R&D Systems
Sourced in United States, Germany
RhCXCL12 is a recombinant human protein that functions as a chemokine. Chemokines are small cytokines that regulate various cellular processes, including cell migration and inflammation. RhCXCL12 is a member of the CXC chemokine family and is the ligand for the CXCR4 receptor.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Pore size polycarbonate filters, by Corning (1 mentions)
Rhcd40, by Abcam (1 mentions)
Anti cxcr4, by R&D Systems (1 mentions)
Rpmi 1640, by R&D Systems (1 mentions)
Matrigel coated nucleopore filter inserts in a 24 well transwell chamber, by Corning (1 mentions)
P paxillin, by Cell Signaling Technology (1 mentions)
Cyclin d1, by Thermo Fisher Scientific (1 mentions)
Ibrutinib, by Selleck Chemicals (1 mentions)
P gsk3β, by Cell Signaling Technology (1 mentions)
Vs 6063, by Selleck Chemicals (1 mentions)
Anti mouse and anti rabbit igg horseradish peroxidase hrp, by Cell Signaling Technology (1 mentions)
P fak, by Cell Signaling Technology (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
C myc, by Abcam (1 mentions)
Phosphate buffered saline (pbs), by Corning (1 mentions)
Superscript first strand synthesis system, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
6 protocols using rhcxcl12
1
T-ALL Cell Migration Assay
2
Chemotaxis Assay for Natural Killer Cells
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Chemotaxis was assessed as previously described.31 Briefly, the upper well of Transwell plates (24‐well, 5.0 μm pore size; Corning, USA) were seeded with purified pNK suspensions (106 cells/200 μl) while the bottom chamber contained TCM (800 μl) with or without anti‐CXCR4 (1 μg/ml; R&D Systems, USA), or control medium with or without rhCXCL12 (R&D Systems, USA). Cells migrating into the lower chamber after 3 h at 37°C were isolated and labelled with fluorescence‐conjugated antibodies. The absolute number of chemotactic cells was determined by FCM as described.31
3
Transwell Invasion Assay with Matrigel
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Matrigel-coated nucleopore filter inserts in a 24-well transwell chamber (Corning Biocoat, New York, USA) were used for the invasion assays. Cells (treated or untreated with VS-6063) were seeded at a density of 40,000 cells/well into the upper part of the Matrigel-coated filter, and RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum and rhCXCL-12 (R&D Systems, Wiesbaden, Germany) (1 ng/mL) was added to the lower part. After 24 h, the cells that had migrated through the Matrigel and the 8-μm pore-size membrane were fixed, stained, and counted under a light microscope.
4
Immunoblotting and Immunohistochemistry Protocol for Cellular Signaling
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The following antibodies were used for immunoblotting and immunohistochemistry: FAK, pFAK (Tyr397), pPaxillin (Tyr118), pAKT (Ser473), actin, p-p42/44 (Tyr202/204), pGSK3β (Ser9), pIκBα (Ser32/36), IKKα, pIKKα/β (Ser176/180), p52, cleaved caspase-3, anti-mouse and anti-rabbit IgG horseradish peroxidase (HRP)-linked from Cell Signaling (Beverly, MA, USA). Cyclin D1 was obtained from Thermo Scientific (Waltham, MA, USA); c-Myc was from Abcam (Cambridge, UK). Immunodetection was performed with the DAKO REAL detection kit (DAKO GmbH, Hamburg, Germany).
The following inhibitors and immunoreagents were used: VS-6063 (Selleckchem, Muenchen, Germany), ibrutinib (Selleckchem, Muenchen, Germany), and rhCXCL-12 (R&D Systems, Wiesbaden, Germany).
The following inhibitors and immunoreagents were used: VS-6063 (Selleckchem, Muenchen, Germany), ibrutinib (Selleckchem, Muenchen, Germany), and rhCXCL-12 (R&D Systems, Wiesbaden, Germany).
5
Transcriptomic Analysis of CXCL12-Treated Endometrial Stromal Cells
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ESCs were purified and cultured from endometrial tissue as previously described (7 (link)). The ESCs exhibited 92% purity (Vimentin positive). ESCs from the same patients were used in control and treatment groups (healthy ESCs and recombinant human (rh)CXCL12-treated healthy ESCs). To clarify the miRNAome and mRNAome of CXCL12-treated ESCs, cells were treated with 100 ng/ml rhCXCL12 (R&D Systems, Inc., Minneapolis, MN, USA) for 48 h, whereas the control ESCs were cultured with 1% phosphate-buffered saline (PBS; Corning Incorporated, Corning, NY, USA) as the vehicle control. The rhCXCL12 treatment dose and exposure time were selected according to our previous study (7 (link)).
6
CXCR4 Transcriptional Regulation by CXCL12
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The expression of CXCR4 mRNA (receptor for CXCL12) was analyzed over a 24 hour period for all three cell lines to determine if exposure to its CXCL12 ligand over time transcriptionally regulates CXCR4 gene expression. 1.5×105 cells were inoculated in 24-well plates in triplicate with or without 100 ng of rhCXCL12 (R&D Systems, Inc; 350-NS-010). After 0, 2, 8, or 24 hours of culture, 1 mL of TRIzol reagent (Invitrogen) was added to lyse the cells and all lysate was collected and frozen in -20 C until further processing.
Purified RNA was obtained using the RNeasy Minikit (QIAGEN) as per the manufacturer’s protocol. The RNA pellets was allowed to dry for 15 min before 20 μL of nuclease-free water was added to each of the samples in preparation for spectroscopy examination (Nanodrop, Thermo Scientific). cDNA was synthesized using the Superscript First Strand Synthesis System (Invitrogen) and used for real-time polymerase chain reaction (PCR) using SYBR Green PCR Master Mix (Applied Biosciences). The following primers: hCXCR4 forward: 5’-AGCATGACGGACAAGTACAGG-3’ and hCXCR4 reverse: 5’-GATGAAGTCGGGAATAGTCAGC-3’ were designed using Primer Express, Version 3.0 software (ABI Prism; PerkinElmer Life and Analytical Sciences) and synthesized by Invitrogen. Data were analyzed according to the comparative threshold method and normalized against the GAPDH internal control transcript.
Purified RNA was obtained using the RNeasy Minikit (QIAGEN) as per the manufacturer’s protocol. The RNA pellets was allowed to dry for 15 min before 20 μL of nuclease-free water was added to each of the samples in preparation for spectroscopy examination (Nanodrop, Thermo Scientific). cDNA was synthesized using the Superscript First Strand Synthesis System (Invitrogen) and used for real-time polymerase chain reaction (PCR) using SYBR Green PCR Master Mix (Applied Biosciences). The following primers: hCXCR4 forward: 5’-AGCATGACGGACAAGTACAGG-3’ and hCXCR4 reverse: 5’-GATGAAGTCGGGAATAGTCAGC-3’ were designed using Primer Express, Version 3.0 software (ABI Prism; PerkinElmer Life and Analytical Sciences) and synthesized by Invitrogen. Data were analyzed according to the comparative threshold method and normalized against the GAPDH internal control transcript.
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