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NEWS

You can now order Cell Biologics' products through Fisher Scientific. Search by Cell Biologics' product catalog number or key words...

 

in April 2017

in May

Phone: 312-226-8198

Fax:      312-226-8958

E-mail: info@cellbiologics.com

 

PROMOTIONS


  • Get Free Gelatin-Based Coating Solution per primary cell order,  which can be used for most primary cell culture from CellBiologics through 2017.

NEW PRODUCTS


Protocol for Flow Cytometry, LDL uptake and Tube Formation Assay

Flow Cytometry In Mouse Primary Endothelial Cell Analysis

  • General protocol for flow cytometry procedure to use an unconjugated primary antibody.
  • Harvest Mouse Endothelial Cells and keep cells in the cell culture medium (M1168, freshly made Medium) containing 1-10 ng/ml VEGF and 10% FCS for 20 min at 37°C. (note: you may let cells over-growing for 24-48h after cells reach confluence; it may take 3-5 days for cells to reach confluence in a T25 flask)
  • Wash the cells 1 time with blocking buffer (1% BSA in 1X PBS with Calcium & Magnesium).
  • Adjust Mouse Endothelial Cell suspension to a concentration of at least 0.5 x 106 cells/ml in 1-2% BSA in 1X PBS with Calcium & Magnesium.
  • Keep cells in blocking buffer for 30 min at RT.
  • Add 0.1-10 μg/ml of the primary antibody (UNCONJUGATED). In general, we use 1.0 ug/ml of anti mouse CD31 antibody, Catalog No. AF3628, Anti-Mouse CD31/PECAM-1 (Polyclonal Goat IgG) from R&D SYSTEMS, INC. or CD31/PECAM-1 (Purified Rat Anti-Mouse CD31, Catalog No. 553370, BD).
  • Take 1.0 ul primary antibody from stock solution (0.2mg/ml) into 200 ul sample.
  • Incubate cells in eppendorf tubes for at least 30-45 min with gentle shaking at RT.
  • Wash the cells two times (1-1.5 ml blocking buffer) by centrifugation at 200 g for 5 minutes and resuspend them in 500 µl.
  • Add Second Antibody, Catalog No.: A-11055 (1:200 - 1:400 dilution, The Alexa Fluor® 488 Donkey Anti-Goat IgG (H+L), Invitrogen) and incubate in the dark at room temperature with gentle shaking for 30 minutes.
  • Wash the cells 2 times (1-1.5 ml blocking buffer) by centrifugation at 200 g for 5 minutes and resuspend them in 0.4 ml (5 ml Round-Bottom Tube with Cell-Strainer Cap, Catalog No. 352235 BD).
  • Keep the cells in the dark on ice and analyze the cells ASAP between 5 min - 2 hours by FACS.
  • You may let cells over-growing for 24-48h after cells reach confluence before doing any cell testing, cell staining, FACS or designed experiments.

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Uptake of Dil-Ac-LDL by Endothelial Cells

  • Cells are seeded in a 24-well plate coated with gelatin-based coating solution (catalog # 6950, Cell Biologics) for 5-10 minutes to 70–90% confluence.
  • Wash cells two times with 1% BSA in 1X PBS with Calcium & Magnesium (wash buffer).
  • Incubate cells with cell culture medium containing 0.05% FBS for 2 hours in an incubator (370C).
  • Remove cell culture medium from the plate.
  • Incubate endothelial cells with Dil-Ac-LDL for 4 h at 37°C. Note, mix Dil-Ac-LDL at the concentration of 10 ug/ml (Catalog No. L-35353/Alexa 594, Life Technologies) with complete cell culture medium containing 2-10 % FBS.
  • Decant the medium and wash the cells 3 times with wash buffer, 3 min each wash.
  • Keep the cells with 0.5-1.0 ml medium in the dark to analyze under a fluorescence microscope ASAP between 5 min - 4 hours (or fix cells and keep cells at 4°C in the dark until the scheduled time for analysis before 12-24 hours).
  • For the best result, analyze the cells as soon as possible after staining procedure.

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Mouse Endothelial Tube Formation Assay (In Vitro Angiogenesis)

Preparing Matrigel:

  • Thaw reduced growth factor matrigel (BD: 354234) by submerging the bottle on ice and store the matrigel at 4 °C overnight.

Preparing Cells:

  • Wash 90-95% confluent endothelial cells with sterile PBS (1X) without calcium and magnesium twice.
  • Incubate cells (on T25 or T75flask) with warm 0.25% Trypsin-EDTA solution (Cell Biologics, Catalog No. 6914) for 3-5 minutes at 37°C. Once cells detach (slightly taps on the flask can be used to speed up the detachment), add 6-10 ml culture medium (Cell Biologics' Cell Culture Medium supplemented with 5-10 % FBS) to the flask to end the digestion (neutralizing the Trypsin).
  • Filter the cell suspension through a sterile 40µm nylon mesh filter (BD 352340) to remove remaining clumps.
  • Count cells and resuspend cells to a final concentration of 2.5-5.0 x 105 cells/ml in endothelial cell culture medium with 50 ng/ml VEGF.

Tube Formation Assay

  • Pipet 200 µl matrigel into each well of a 24-well plate. Avoid creating bubbles.
  • Incubate the 24-well plate at 37 °C and 5% CO2 for 30 min to solidify matrigel.
  • Once matrigel has set, seed 50,000 cells -100,000(200ul) into each well.
  • Observe the tube formation after 4-6 hrs. Peak tube formation may occur between 12hrs and 24hrs. 
  • Take image by 4X lenses microscope. 

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Protocol for immunofluorescence staining

·        Cells are seeded in a 24-well plate coated with gelatin-based coating solution (catalog # 6950, Cell Biologics) for 5-10 minutes to 90–100% confluence.

·        Wash cells two times with 1% BSA in 1X PBS with Calcium & Magnesium (wash buffer).

·        Fix cells 3.7% Formaldehyde (Dilute 37% stock in Blocking buffer) 20min at RT.

·        Remobilize cells (if need be) in Blocking buffer (0.4% Triton X 100, 1%BSA in 1xPBS) 20min at RT.

·        Add first antibody1:100-1:1000 in blocking buffer at 40C overnight, and humidifier.

·        Wash the cells three times by using 1xPBS.

·        Second antibody 1:200 in blocking buffer, 1hr, RT, DARK, humidifier, and DAPI if needed.

·        Wash the cells three times by using 1xPBS.

·        Keep the cells with 0.5-1.0 ml 1xPBS in the dark to analyze under a fluorescence microscope or Mount with Prolong.

·        For the best result, analyze the cells as soon as possible after staining procedure.

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Note:

·        Adjust the volume of endothelial culture medium and number of cells plated depending on the type of endothelial cell used.

·        Please send us the cell images (>90% confluence) if you have any questions or problems with cultured cells.

 

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