Bone marrow cells were lineage (B220−Mac-1−Gr-1−CD16/32−) depleted on an AutoMACS Pro system (Miltenyi) and suspended in StemSpan SFEM (StemCell Technologies) supplemented with 2.6% fetal bovine serum, 1% L-glutamine and stem cell factor (20 ng/mL) then cultured for 2 days at 37°C in 5% CO2. Cells were transferred into fresh medium containing thrombopoietin (50 ng/mL) and cultured for 4 more days. Mature megakaryocytes were enriched on a bovine serum albumin gradient as previously described15 (link) and plated in 12-well μ-Chamber glass slides (ibidi) coated with fibronectin (500 μg/mL; Life Technology), fibrinogen (100 μg/mL; Hyphen BioMed) or collagen (1:20; StemCell Technologies). Cells were then allowed to spread for 3–8 h. Cells were fixed in μ-Chamber slides with 4% formaldehyde/phosphate-buffered saline, permeabilized with 0.1% Triton-X100/phosphate-buffered saline and blocked with FcBlock (1:500; BD Biosciences), then labeled with FITC-CD41 (BD Biosciences) and AF555-β-tubulin (Cell Signaling) antibodies or AF555-Phalloidin (Molecular Probes). All conditions for both controls and knockouts were applied to the same μ-Chamber slide to minimize variation between samples.
Af555 β tubulin
Manufactured by Cell Signaling Technology
AF555-β-tubulin is a fluorescently labeled tubulin protein that is used in cell biology research. It is conjugated to the Alexa Fluor 555 dye, which emits red fluorescence when excited by appropriate wavelengths of light. This product can be used to visualize and study the microtubule cytoskeleton within cells.
Automatically generated - may contain errors
Lab products found in correlation
Fibronectin, by Thermo Fisher Scientific (2 mentions)
Cd41 fitc, by BD (2 mentions)
Fc block, by BD (2 mentions)
Fibrinogen, by Hyphen Biomed (1 mentions)
Automacs pro system, by Miltenyi Biotec (1 mentions)
Stemspan sfem, by STEMCELL (1 mentions)
Vectorshield containing dapi, by Vector Laboratories (1 mentions)
Leitz dmrb microscope, by Leica (1 mentions)
Wnt3a, by R&D Systems (1 mentions)
2 protocols using af555 β tubulin
1
Megakaryocyte Spreading Assay
2
Megakaryocyte Differentiation Assay
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Lineage depleted bone marrow from PF4-Cre Gfi1b-flox/flox and Gfi1b-wt/flox mice were cultured in Stemspan/2.6% FBS supplemented with 1% L-Glutamine and SCF (20 ng per ml). TPO (50 ng per ml) was added to fresh medium at day 2 and cells were cultured for 4 more days. MKs were enriched on a BSA gradient and plated on fibronectin (500 µg per ml; Life Technology) coated 12-µ-chamber slide (ibidi). Cells were incubated at 37 °C, 5% CO2 for 3–6 h with or without Wnt3A (R&D systems) allowing megakaryocytes to attach and spread. After incubation cells were fixed with 4% formalin, permeabilized with 0.1% Triton-X100 in PBS, and blocked with FcBlock (1:500; BD Biosciences). Cells were then labeled with FITC-CD41 (BD Biosciences), AF555-β-tubulin (Cell Signaling) and covered with Vector Shield containing DAPI (Vector Laboratories). Immunofluorescence imaging of the slides was done using a DCX-950P DP72 camera (Sony) mounted on a Leitz DMRB microscope (Leica). Images were analyzed using ImageJ v1.46r (NIH).
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