General Protocol for Recovering or Freezing Primary Cells

All cell culture procedures must be conducted in a bio-safety cabinet.

Any and all media, supplements, and reagents must be sterilized by filtration through a 0.2 µm filter.

Use aseptic technique to prevent microbial contamination.

Cryo-preserved cells must be stored in liquid nitrogen or seeded immediately upon arrival.

Medium:

Review the information provided on the Cell Biologics website about appropriate culture media (e.g. serum and other supplements). Use pre-warmed (37°C) cell culture media (30-50 ML) to recover cryo-preserved cells and when changing media or splitting cells.

Coating of flasks or dishes:

Coat sterile culture dishes or flasks with Gelatin-Based Coating Solution (Cell Biologics, Catalog No. 6950) for 10-30 min and then aspirate the excess solution before seeding cells.

Cell recovery from cryovial:

• Quickly thaw cells in cryo-vial by incubating them in a 37°C water bath for <1 min until there is just a small bit of ice left in the vial.
• Promptly remove the vial and wipe it down with 70% ethanol.
• Transfer cells from the vial to a sterile centrifuge tube. Add 8-10 ml of pre-warmed Cell Biologics Cell Culture Medium.
• Flush the vial with an additional 0.5-1 ml of medium to ensure complete transfer of cells to the centrifuge tube.
• Centrifuge cells at 200 g for 5 minutes.
• Aspirate the supernatant and resuspend the cell pellet in 6 ml of Cell Biologics’ Cell Culture Growth Medium.
• Add resuspended cells into a T25 flask pre-coated with Gelatin-Based Coating Solution (Cell Biologics, Catalog No. 6950).
• Place the T25 flask in a humidified, 5%-CO₂ incubator at 37°C.
• Change culture media the following day to remove non-adherent cells and replenish nutrients.
• Change cell culture medium everyday when cells are >70% confluent.
• Cells should be checked daily under a microscope to verify appropriate cell morphology.

Expansion of cultured primary cells:

• Remove and discard the cell culture media from the flask.
• Flush the adherent layer 2 times using a 5 ml sterile pipette with sterile PBS (1X) without calcium and magnesium to dislodge loosely attached cells and remove fraction.
• Remove and discard the wash solution from the flask.
• Incubate cells with warm (37°C) 0.05% or 0.25% Trypsin-EDTA solution (Cell Biologics, Catalog No. 6914) for 3-5 minutes. Use 3.0 ml of Trypsin-EDTA solution when collecting cells from T75 flasks, and 2 ml when using T25 flasks. As soon as cells have detached (the flask may require a few firm gentle taps), add 8-10 ml of Cell Biologics’ Cell Culture Medium supplemented with 5-10 % FBS to a T25 or T75 flask (the FBS will neutralize the trypsin).
• Plate cells in fresh flasks or plates precoated with Gelatin-Based Coating Solution in a humidified, 5%-CO₂ incubator at 37°C.
• Change culture media the following day to remove non-adherent cells and replenish nutrients.
• Cells should be checked daily under a microscopy to verify appropriate cell morphology.
• Change culture medium every 24-48 hours. Please note that the medium should be changed every day when cells are >70% confluent to remove non-adherent cells and replenish nutrients. Pre-wash cells with 1X PBS 1-2 times whenever replacing the medium.

We recommend splitting primary cells at the follow ratio:

• The recommended split ratio for primary murine cells is 1:2.
• A confluent monolayer of primary cells grown in a T75 flask may be expanded on a 6-well plate ready for use in experiments under the cell culture conditions specified by Cell Biologics.

Procedure for Freezing Cells

Materials:

• 1X Phosphate Buffered Saline (PBS-1X)
• 0.05% or 0.25% Trypsin-EDTA (1X) solution (Cell Biologics, Catalog No. 6914)
• Tissue Culture Media
• Cold Freezing Media (Cell Biologics, Catalog No. 6916).
• Labeled Cryovials
• Confluent cells

------------------------------------------------------------------

• Remove and discard the cell culture media from the flask.
• Flush the adherent layer with a 5 ml sterile pipette 2 times with sterile PBS (1X) without calcium and magnesium to dislodge loosely attached cells and remove fraction.
• Remove and discard the wash solution from the flask.
• Incubate cells with warm (37°C) 0.05% or 0.25% Trypsin-EDTA solution (Cell Biologics, Catalog No. 6914) for 3-5 minutes. Use 3.0 ml of 0.25% Trypsin-EDTA solution when collecting cells from T75 flasks, and 2 ml when using T25 flasks. As soon as cells have detached (the flask may require a few firm gentle taps), add 10 ml of Cell Culture Medium supplemented with 5-10 % FBS to the flask (the FBS will neutralize the trypsin).
• Centrifuge the cell suspension at 200 g for 5 minutes.
• Remove supernatant with sterile Pasteur pipette.
• Quickly re-suspend pellet by adding 1 ml freezing media per vial to be frozen.
• Place vials in Nalgene "Mr. Frosty" freezing container containing100% isopropyl alcohol at -70-80 °C for 24 h.
• Transfer vials to liquid N₂ tank for indefinite storage.

We recommend freezing primary cells at the follow ratio:

• A confluent primary endothelial cells grown in a T75 flask may be frozen in 2 cryovials.
• A confluent primary endothelial cells grown in a T25 flask may be frozen in 1 cryovial.

Flow Cytometry In Mouse Primary Endothelial Cell Analysis

General protocol for flow cytometry procedure to use an unconjugated primary antibody (2-step staining)

• Harvest Mouse Endothelial Cells (note: you may let cells over-growing for 24-48h after cells reach confluence; it may take 3-4 days for cell to reach confluence in a T25 flask) and keep cells in the cell culture medium (M1168, freshly made Medium, 1-2 weeks) containing 1-10 ng/ml VEGF and 10% FCS for 20 min at 37°C.
• 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.

 

GFP Cell Preparation

·       GFP-Labeled Primary Cells employing Amaxa nucleofector transfection methods are detached from the culture flask and immediately cryo-preserved in vials. Each vial contains 0.5-1.0x10⁶ cells per ml and is delivered frozen. GFP marked primary cells are verified by a fluorescent microscope.

·       Cells can be seeded on 30-60 mm culture dishes ready for experiments (or cells may be expanded for 1-2 passages at a split ratio of 1:2) under the cell culture conditions specified by Cell Biologics. Repeated freezing and thawing of cells is not recommended.

Protocols for GFP-labeled Primary mammalian cells   

·       Harvest the cells by trypsinization. Count an aliquot of the trypsinized cells and determine cell density.

·       Centrifuge the required number of cells (0.5-1.5 x10⁶ cells per sample) at 220xg for 5 minutes at room temperature.

·       Resuspend the cell pellet carefully in 100 µl room temperature Nucleofector™ Solution per sample.

·       Combine 100 µl of cell suspension with 5-10 µg pmaxGFP™ Vector (recommended for initial optimization).

·       Transfer cell/DNA suspension into certified cuvette; sample must cover the bottom of the cuvette without air bubbles. Close the cuvette with the cap.

·       Select the appropriate Nucleofector™ Program.

·       Insert the cuvette with cell/DNA suspension into the Nucleofector™ Cuvette Holder and apply the selected program.

·       Take the cuvette out of the holder once the program is finished.

·       Add 500 µl of the pre-equilibrated culture media to the cuvette and gently transfer the sample immediately into the culture dish. Use the supplied pipettes and avoid repeated aspiration of the sample.

·       Incubate the cells in a humidified 37°C/5% CO₂ incubator until analysis. GFP expression is often detectable after only 4 – 8 hours but ideally, cells should be left undisturbed for 24 hours. GFP expression efficiency depends on different cell types.

Transfection efficiencies: ~40-60% by using Amaxa's Nucleofector device