Monthly Archives: March 2018

Ficoll 400

Overview:

Synonyms: Ficoll 400, Polysucrose-400, Ficoll PM 400
CAS Number: 26873-85-8
Chemical structure: (C12H20O10)n
Molecular Weight: ≈400,000

Appearance: White to yellowish powder

  • Ficoll 400 is high molecular weight non-ionic, highly branched, a synthetic polymer of sucrose. It is synthesized by the copolymerization of sucrose and epichlorohydrin.
  • Ficoll 400 is hydrophilic. It dissolves readily in water due to the high content of hydroxyl groups. Depending on the concentration, It forms clear to slightly hazy, colorless to yellowish solution in water.
  • The aqueous solution of Ficoll 400 is stable at alkaline and neutral pH but rapidly hydrolyzed at pH below 3.0, particularly at elevated temperatures.
  • Comparatively low osmolality and low membrane permeability make Ficoll 400 a better choice than sucrose for biological applications particularly for the separation of cells.

Applications:

  • Isolation and purification of cells, cell membranes, cell organelles and viruses.
  • Denhardt’s solution, which is used for Northern and Southern hybridization, contains Ficoll 400.
  • Ficoll 400 can also be used as a high density agent for the preparation of DNA/RNA loading dye for agarose and polyacrylamide gel electrophoresis of DNA/RNA

Suppliers – Ficoll 400

Overview:

CAS Number: 26873-85-8
EC Number: 200-334-9
Molecular Weight: ≈400,000 g/mol

Synonyms: Ficoll 400, Polysucrose-400

Ficoll 400 can be purchased from the following suppliers. Please visit supplier’s product page for more information.

From Merck Millipore, Catalog No. 4550-OP

Product page ‖‖‖ Product Information Sheet ‖‖‖

From Merck Millipore, Catalog No. 341691

Product page ‖‖‖ Product Information Sheet ‖‖‖

From Sigma-Aldrich, Catalog No. F4375

Product page ‖‖‖ Product Information Sheet ‖‖‖

From Sigma-Aldrich, Catalog No. 341691

Product page ‖‖‖ Product Information Sheet ‖‖‖

From abcam, Catalog No. ab146570

Product page ‖‖‖ Product Information Sheet ‖‖‖

From Applichem, Catalog No. A2252  

Product page ‖‖‖ Product Information Sheet ‖‖‖

From Fisher Scientific, Brand- Fisher Bioreagents BP525-10
Catalog No. 10295003 (10g), 10071593 (25g), 10468343 (100g), 10478343 (500g)

Product page ‖‖‖ Product Information Sheet ‖‖‖

From GE Life Sciences, Catalog No. 17030010 (100g), 17030050 (500g), 17030005 (5kg), 17030008 (40kg)

Product page ‖‖‖ Product Information Sheet ‖‖‖

From VWR Life Science, Catalog No. 437092S (25g), 437093T (250g)
Molecular biology grade

Product page ‖‖‖ Product Information Sheet ‖‖‖

Suppliers – Xylene cyanol FF

Overview:

Molecular Formula: C25H27N2NaO6S2
CAS Number: 2650-17-1
EC Number: 220-167-5
Molecular Weight: 538.61

Synonyms: Acid blue 147; Cyanol FF, XC; Xylene Cyanole FF

Xylene cyanol FF can be purchased from the following suppliers. Please visit supplier’s product page for more information.

From Merck Millipore, Catalog No. 9710-OP

Product page ‖‖‖ Product Information Sheet ‖‖‖

From Sigma-Aldrich, Catalog No. X4126

Product page ‖‖‖ Product Information Sheet ‖‖‖

From abcam, Catalog No. ab146296

Product page ‖‖‖ Product Information Sheet ‖‖‖

From Applichem, Catalog No. A4976   

Product page ‖‖‖ Product Information Sheet ‖‖‖

From Fisher Scientific, Catalog No. BP125-100

Product page ‖‖‖ Product Information Sheet ‖‖‖

From Santa Cruz Biotechnology, Catalog No.  sc-206012

Product page ‖‖‖ Product Information Sheet ‖‖‖

Suppliers – Bromophenol Blue

Overview:

Molecular Formula: C₁₉H₁₀Br₄O₅S
EC Number: 204-086-2
CAS Number: 115-39-9
Molar Mass: 669.96 g/mol

Synonyms: Bromophenol Blue, 3,3′,5,5′-Tetrabromophenolsulfonphthalein, BPB

Bromophenol Blue can be purchased from the following suppliers. Please visit supplier’s product page for more information.

From Merck Millipore, Catalog No. 108122

Product page ‖‖‖ Product Information Sheet ‖‖‖

From Sigma-Aldrich, Catalog No. B0126

Product page ‖‖‖ Product Information Sheet ‖‖‖

From abcam, Catalog No. ab146339

Product page ‖‖‖ Product Information Sheet ‖‖‖

From Applichem, Catalog No. A2331   

Product page  ‖‖‖ Product Information Sheet ‖‖‖

From Fisher Scientific, Catalog No. B392-5

Product page ‖‖‖ Product Information Sheet ‖‖‖

From Santa Cruz Biotechnology, Catalog No.  sc-24971

Product page  ‖‖‖ Product Information Sheet ‖‖‖ 

Passaging/subculturing methods for cell culture

  • Several methods have been developed for passaging cells. Each method has its own advantages and drawbacks. Always check the cell line instruction manual and relevant literature for the optimal procedure.
  • Most passaging methods aim to obtain single cell suspension by breaking cell-substratum and cell-cell contacts. The single cell suspension is further diluted by addition of fresh growth medium and allowed to grow in its optimal growth environment.
  • Depending on the experimental requirement, type and growth mode of cell culture, and nature of cell line, one should choose a particular method.
  • Depending on growth mode, cell culture can be adherent, semi-adherent and non-adherent. Since nonadherent cells grow in suspension, such culture can be passaged essentially by diluting the existing cell culture. In contrast, semi-adherent and firmly adherent cells need to be detached from the substratum for passaging.
  • Several methods have been developed for passaging cell culture. These methods can be broadly classified into three types based on the nature of treatment used to dissociate cells.
    • Enzymatic methods (e.g., Trypsin, Collagenase, Dispase)
    • Mechanical methods (Scraping by rubber policeman, Dislodging cells by vigorous shaking)
    • Treatment with chelators (e.g., EDTA or EGTA treatment)
  • Most commonly used method, the Trypsin-EDTA method for all regular adherent cell culture, combines both enzymatic and chelator treatment.
Enzymatic treatments (e.g., Trypsin, Collagenase, Dispase):
  • Enzymatic methods are routinely used in most cell culture laboratories for passaging adherent cells.
  • A brief treatment of cells with enzyme solution and subsequent gentle pipetting results in single cell suspension.
  • Enzymatic methods are very efficient at disrupting both cell-substratum as well as cell-cell contacts, leading to single cell suspension. Cell number can be determined easily in single cell suspension, therefore, enzymatic methods are valuable for those experiments where accurate cell number is one of the requirements of experimental procedure. However, enzymatic treatments results in digestion and cleavage of cell surface proteins (e.g., receptors), therefore, not suitable for experiments which aims to analyze cell surface proteins.
  • The sensitivity of cell lines for a particular enzymatic method may differ. Therefore, one needs to standardize treatment condition. Overtreatment with enzyme solution can lead to cell death, whereas, under treatment can cause inefficient detachment of cells and lead to cell clumps.
  • Often inhibitors (e.g., serum, soybean trypsin inhibitor etc.) are added to stop the enzyme action.
Mechanical means (Scraping by rubber policeman, Dislodging semi-adherent cells by vigorous shaking):
  • Mechanical means involved disrupting cell-substratum interaction by mechanical force like scraping using rubber policeman or vigorously tapping the culture dish or extensively pipetting culture medium over cell layer.
  • This method often leads to cell death and is not recommended for tightly adherent cells.
  • Cell clumps are often observed therefore determining cell number using haemocytometer counting is difficult. However, this method is suitable for semi-adherent cells.
Treatment with chelators (e.g., EDTA or EGTA treatment)
  • Chelators sequester the divalent cations, e.g., Ca2+ or Mg2+, which are required for adhesion of cell to substratum as well as cell-cell contacts.
  • This method takes long time incubation with chelator solution. Most cell lines partially loses their contact with substratum and subsequently extensive pipetting or tapping of culture dishes detaches cells from the substratum.

Protocol – Subculturing suspension cells

Protocol – Subculturing suspension cells

Overview:

  • Suspension cell cultures are passaged by diluting the existing culture.
  • Since cells float in the medium in suspension culture, they are not treated with trypsin-EDTA solution unless there are some special requirements. For example, if you need to plate estimated number of cells from a culture which forms tight clumps in suspension. In this case, cell clumps are treated with trypsin-EDTA to obtain single cell suspension to count the cell number accurately.
  • To subculture suspension cells, a small amount of cell suspension from the existing culture is transferred to a culture dish containing fresh growth medium.

Requirements:

  • Reagents and solutions:
    • Complete growth medium (room temperature / 37°C)
  • Equipment and disposables
    • T25 flask/Tissue culture dishes
    • Pipettes and pipette aid
    • Laminar flow hood
    • Beaker to discard the waste
Starting material:

Suspension cell culture (high density) ready to subculture

Prior to start:
  • Place complete medium in 37°C water bath for warming.
  • Clean and wipe workspace in the laminar flow hood with 70% ethanol, turn on UV light for 20 – 30 min. After 20 min, turn off the UV light and start the air flow. Let it flow for 10 min.

Objective:

Subculturing of suspension culture growing

Note:
  • Check cells under the microscope to make sure cells are healthy and are not contaminated.
  • Use aseptic techniques while operating cell culture.

Procedure:

Step 1: Transfer all reagent bottles and disposables to the laminar flow hood
  • Spray and wipe all bottles and packets containing disposables (culture dish packets) with 70% ethanol and place them in the laminar flow hood.
  • Take out the estimated number of culture dishes from its packet and label them with the date of subculture, passage number, cell name and your name.
Step 2: Dilute cell suspension at recommended cell density (or split ratio)
  • Take out culture dish from the incubator and place inside laminar flow hood.
  • Pipet suspensension culture to suspend cells homogeneously and transfer an aliquot of cells into fresh culture flask/dish.
  • Add appropriate amount of growth medium (for T25 flask/ 60 mm dish, 4 – 5 ml medium is recommended).
  • Pipette up and down 2 – 3 times to suspend cells homogeneously.
  • Wright all details on culture dish including your name, cell line name, passage number and subculture date.
  • Discard remaining cell suspension if you don’t need them.
Note:
  • Cell counting is not necessary for the regular maintenance of cell in culture. Most researchers use split ratio to seed cells in a fresh culture dish.
  • Split ratio is a rough estimation of how many culture dishes can be prepared from the existing cell culture. For e.g., you can prepare 5 to 10 flask/dish from the cell culture (90% confluent) if the recommended split ratio is 1:5 to 1:10.
Tips:
  • If recommended split ratio is 1:5, transfer ⅕th culture volume to the fresh culture dish. Alternatively, cells can be counted using haemocytometer or any other appropriate method and recommended number of cells can be transferred to the fresh dish. Often counting is performed to seed exact number of cells as needed for the experiment.
  • To count cells accurately, cell clumps should to broken into the single cells. To make single cell suspension, try several rounds of gentle pipetting. If still cell clumps are seen, collect cells by centrifugation, wash the pellet with PBS and briefly treat cells with trypsin-EDTA.
  • If you need to make many culture dishes/flasks, prepare master mix before transferring suspension to the fresh flask.

Step 3: Place the flask in the incubator. Open the lid of the flask slightly for air exchange if you are using sodium bicarbonate containing culture medium. Tighten the lid if you are using vented cap flask.

Protocol – Subculturing adherent cells growing in the serum-containing medium using Trypsin-EDTA

Overview:

  • Trypsin-EDTA method also referred to as trypsinization, is a most commonly used method for passaging/subculturing adherent cells.
  • Cells to be subcultured are first washed with Ca2+-free and Mg2+-free PBS and subsequently treated with Trypsin-EDTA solution.
  • Brief incubation with Trypsin-EDTA disrupts cell-cell and cell-substratum interactions, resulting in the single cell suspension.
  • Trypsin, a proteolytic enzyme, disrupts these interactions by proteolysis and EDTA by chelating divalent cations.
  • Single cell suspension can be used for the determination of cell number and preparation of fresh culture.

Requirements:

  • Reagents and solutions:
    • 1 X Trypsin-EDTA solution (Room Temperature / 37°C)
    • PBS (Ca2+-free and Mg2+-free) (Room Temperature / 37°C)
    • Complete growth medium (Room Temperature / 37°C)
  • Equipment and disposables
    • T25 flask/Tissue culture dishes
    • Pipette and pipette aid (Glass Pipettes)
    • Laminar flow hood
    • Beaker to discard the waste

Starting material: 90% confluent T25 flask/60 mm dish

Prior to start:
  • Place all reagent bottles – Trypsin EDTA, PBS and complete medium in 37°C water bath.
  • Wipe laminar flow hood with 70% ethanol, turn on UV light for 20 – 30 min. After 20 min, turn off the UV light and start the air flow. Let the air flow for 10 min before use.
  • Now wipe the surface of bottles of all reagents and place them in the laminar flow hood.
  • Properly label all dishes/flask with the date of subculture, passage number, cell name and your name.
  • Check cells under the microscope to make sure cells are healthy and are not contaminated.

Objective:

Subculturing of subconfluent (90% confluent) culture growing in a T25 flask/60 mm culture dish

Precautions:
  • Do all operations aseptically.
  • All transfer of medium should be done inside the laminar flow hood.
  • Wear lab coat and disposable latex gloves at all times.
  • Do all steps which involve changing medium from cells quickly to avoid any risk of drying of cells.
  • Don’t decant medium from the flask/dish. This can increase the risk of contamination.

Procedure:

Step 1: Remove and discard culture medium from the flask and wash cells with PBS free of Ca2+ and Mg2+.
  • Take out the flask from the incubator and place inside laminar flow hood.
  • Slightly tilt the flask/dish. All medium will be collected at tilted side. Now remove and discard all medium.
  • Add 3 – 4 ml PBS. Swirl or tilt the dish/flask in opposite direction 2-3 times. Remove and discard PBS.
Note:
  • Serum in the culture medium has trypsin inactivating activity.
  • PBS washing will remove dead cells, cell debris and remaining growth medium.
Tips:
  • To remove liquid from the flask/dish, you can use vacuum aspirator. Vacuum aspirator is very quick and convenient way to remove liquid from the dish. If you don’t have Vacuum aspirator, use pipette and pipette aid to remove liquids from the flask.
  • Always keep the flow of PBS on side walls of the culture vessel. This will minimize the cell detachment due to flow of the liquid.
Precautions:
  • While pipetting PBS into the flask, take care that the flow of PBS should not disturb cell monolayer.
  • Some cell lines are loosely adherent. In such case, care must be taken to avoid any loss while removing culture medium and washing with PBS.
Step 2: Add Trypsin – EDTA solution and incubate 1 – 5 min at 37°C.
  • Add 1 ml Trypsin-EDTA solution and gently spread trypsin solution all over the cell surface by swirling/tilting the vessel . Incubate at 37°C (in incubator) until cells appear rounded and start detaching from the substratum (it can take 1-5 min for most of the cell lines).
  • Gently tap the flask to remove all cells from the surface.
Notes:
  • 0.5 – 1 ml Trypsin-EDTA solution is sufficient for T25 flask/60 mm dish. Depending on the convenience, one can add more trypsin-EDTA solution. The amount of Trypsin-EDTA should be sufficient to cover all the cell surface of the culture vessel.
  • For each cell line, one should optimize trypsin-EDTA treatment condition (incubation time and concentration). Some cell types are strongly adherent and require a higher concentration of the trypsin-EDTA solution as well as longer incubation time.
Tip:
  • Examine the cell morphology under the microscope every minute if you don’t have any idea how long trypsin-EDTA treatment is required for your specific cell line.
Precautions:
  • Make sure trypsin – EDTA solution covers all cell surface. Each cell should come in contact with trypsin solution. Incomplete trypsin treatment may result in cell clumps.
  • Right trypsin-EDTA treatment condition is crucial for successful trypsinization process. While overtreatment will cause cell death, undertreatment results in cell clump and undetached cells in the culture dish.
Step 3: Inactivate trypsin by adding fresh serum containing medium
  • Wash out all the cells from the surface by pipetting the fresh culture medium (4 ml) all over the surface.
  • Disperse all cell clumps by pipetting 2 – 3 times.
Note:
  • Serum has trypsin inactivating activity. If you are using serum-free medium, inactivate trypsin by adding other trypsin inhibitors e.g., soybean trypsin inhibitor.
Tips:
  • While transferring the medium in the vessel, keep the flow of medium toward the surface where cells were attached.
  • If there are cell clumps, disperse them by several rounds of pipetting. More pipetting can cause cell death!
Step 4 (optional): Determine cell number
  • Transfer all cell suspension to a centrifuge tube. Collect cells by centrifugation.
  • Resuspend cells an appropriate amount of complete medium (5 ml) and determine cell number using haemocytometer or any other method.
Notes:
  • Cell counting is not necessary for the regular maintenance of cell culture. Most researchers use split ratio to seed cells in a fresh culture dish.
  • Split ratio is a rough estimation of how many culture dishes can be prepared from the existing cell culture. For e.g., you can prepare 5 to 10 flask/dish from the cell culture (90% confluent) if the split ratio is 1:5 to 1:10.
Step 5: Prepare fresh culture dish from the cell suspension
  • Transfer an aliquot of cells into fresh culture flask/dish.
  • Add fresh growth medium. Pipette cells to resuspend cells again. Wright all details including your name, cell line name, passage number and subculture date.
  • Discard remaining cell suspension if you don’t need them.
  • Keep the flask in the incubator. Open the lid of the flask slightly for air exchange if you are using sodium bicarbonate containing culture medium. Tighten the lid if you are using vented cap flask.
Note:
  • Use recommended seeding density or split ratio to decide how many cells should be transferred to fresh dish/flask. Generally 1:5 – 1:10 split ratio works well for fast-growing cell lines.
Tip:
  • If you need to make many culture dishes/flasks, prepare master mix before transferring suspension to the fresh flask.