VLA-4 clustering assays were performed as described elsewhere, 11 (link) using 7.5 mg/mL VCAM-1/Fc. AML cells were pretreated for 10 min with 10 mg/mL HA, 60 min with 1 mM midostaurin, 30 min with 10 mM PP2 and 30 min with 10 mM cobimetinib (APExBIO, Houston, USA), where indicated. Cells were allowed to adhere for 30 min at 37°C before fixation with 4% paraformaldehyde. Slides were stained with αCD49d (clone AHP1225), αCD29 (clone 12G10) primary antibodies or isotype control (not shown) followed by a secondary antibody. For CD49d cluster analysis of normal progenitor cells from patients with non-myeloid malignancies, cells were additionally stained with αCD34 antibody (clone QBEND-10). For quantification, high-resolution images were acquired on a Leica TCS SP5 II laserscanning microscope using a 63×/1.4-NA oil-immersion objective (Leica, Wetzlar, Germany). The number of clusters was analyzed using ImageJ software by particle analysis setting the size of the particle at >2 pixels.12 (link)
Tcs sp5 2 laser scanning microscope
Manufactured by Leica camera
Sourced in Germany
The Leica TCS SP5 II is a laser scanning microscope designed for high-performance imaging. It features a modular architecture that allows for configuration of various detection channels and laser lines to meet the specific needs of the user's application. The core function of the TCS SP5 II is to provide high-resolution, multicolor imaging capabilities for a wide range of samples.
Automatically generated - may contain errors
Lab products found in correlation
Na oil immersion objective, by Leica camera (4 mentions)
Cobimetinib, by Apexbio (1 mentions)
Alexa fluor labeled secondary antibody, by Thermo Fisher Scientific (1 mentions)
Dabco, by Merck Group (1 mentions)
Human serum albumin (hsa), by Merck Group (1 mentions)
Ix81 microscope, by Olympus (1 mentions)
Vcam 1, by R&D Systems (1 mentions)
4 protocols using tcs sp5 2 laser scanning microscope
1
VLA-4 Clustering in Leukemia Cells
2
Imaging Jurkat Cell Signaling
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Jurkat P116, Jurkat P116 ZAP70-GFP, or Jurkat P116 cells transiently transfected with ZAP70-K369R-GFP were placed on poly-L-lysine coated coverslips and left untreated or pre-treated with 50 μg/ml piceatannol or DMSO for 1 h and subsequently stimulated for 10 min with 0.5 μg/ml CCL19 or CCL21. Cells were fixed in 4% PFA and immunostaining for CD11a was done as described above, but omitting the permeabilization step. Confocal images were acquired on a Leica TCS SP5 II laser scanning microscope using a 63x/1.4 NA oil-immersion objective (Leica).
3
Antigen Presentation and T Cell Imaging
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LK35.2 APCs or mature murine BMDCs were loaded with 10 μM HEL34−45 or 10 μM OVA323−339 antigen for 1 h, washed and placed on poly-L-lysine coated coverslips. 30 min later 3B11 or DO11.10 T cells stably expressing CCR7-YFP were added. Cells were left unstimulated or stimulated with 0.5 μg/ml CCL19 or CCL21 for 1 h. Cells were fixed in 4% PFA for 10 min at room temperature. For staining, cells were permeabilized and incubated with the appropriate primary antibody, followed by incubation with AlexaFluor-labeled secondary antibodies (Life Technologies). Coverslips were mounted using polyvinyl alcohol mounting medium with DABCO (Sigma-Aldrich). Confocal mages were acquired on a Leica TCS SP5 II laser scanning microscope using a 63x/1.4 NA oil-immersion objective (Leica). 3D reconstitution was performed by Huygens software using confocal images with z-step sizes of 0.08 μm. Quantification of co-localization was done by calculating the Pearson's correlation coefficient using ImageJ.
4
CLL Cell Adhesion Assay with VCAM-1
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Falcon culture slides were coated with 7.5 µg/ml VCAM-1 (R&D Systems) overnight, washed, and blocked with 2% human serum albumin (Merck Millipore). Purified CLL cells were incubated for 1 h with or without 1 µM ibrutinib, added to the slides, and allowed to adhere for 30 min at 37°C in the presence or not of 5 µg/ml F(ab)2 anti-IgM before fixation with 4% paraformaldehyde. Slides were stained with primary anti-CD49d antibody (clone AHP1225; Bio-Rad) and Cy3-conjugated secondary anti–rabbit antibody. Images were taken with an Olympus IX81 microscope. For quantification, high-resolution images for cluster analysis were acquired on a Leica TCS SP5 II laser scanning microscope using a 63×/1.4-NA oil-immersion objective (Leica), and the number of clusters was analyzed using ImageJ software.
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