In vitro Foam Cell Formation

In vitro Foam Cell Formation

PURPOSE
Collect baseline macrophages from mice and plate on chamber slides, at a concentration of 600,000 cells/well. The cells will be stimulated the next day with oxLDL at different concentrations of 50μg/ml, 100μg/ml and 150μg/ml for 48 hours. After incubation, the cells will be stained with Oil Red O for lipids, which will demonstrate foam cell formation in these cells, as well as any differences that may be due to their genotype.

MATERIALS

• Macrophage media: 20% FBS in DMEM high-glucose + penicillin:
  • 50ml Fetal Bovine Serum (ATCC Cat. No. 30-2020), thaw at 37°C before adding (20%FBS)
  • 2.5ml Penicillin-Streptomycin (Invitrogen 15140-122), thaw at 37°C before adding (1%)
  • 197.5ml DMEM high-glucose, +L-glu, no Na-pyruvate (GibcoBRL Cat. No. 11965-092). Thaw at 37°C before adding
  • Combine all and filter. Store at 4°C.
• Stimulating media: 10%/FBS DMEM high-glucose + penicillin (prepared same as above):
  • 25ml Fetal Bovine Serum  (10%)
  • 2.5ml Penicillin-Streptomycin (1%)
  • 222.5ml DMEM high-glucose, +L-glu, no Na-pyruvate (GibcoBRL Cat. No. 11965-092). Thaw at 37°C before adding
  • Combine all and filter. Store at 4°C.

Note: Can use FBS or Pen-Strep from any company, as long as it has been kept sterile and you use it consistently throughout the experiment.

• Ice bucket
• 6-well plates for tissue culture
• Mice:
  • All procedures will be performed in the tissue culture hoods, following tissue culture preventive procedures
  • Mice injected with 1.5ml of 4% Thioglycollate 4 days prior.
  • Record all information of mice:
    

    Cage	Mouse	ID	Sex	DOB	Genotype
    WT					+/+
    WT					+/+
    HET					-/+
    HET					-/+

PROTOCOL
Macrophage Extraction: Follow standard IP Lavage Technique

Plating

1. Collect cells
   • Pour cell suspension in 50ml collection tubes.
   • You may pool suspensions of different mice with same genotype.
   • Centrifuge cells at 1K for 10 minutes. Make sure you balance the centrifuge.
   • Discard supernatant.
2. Re-suspend cells
   • Using 10ml pipette, add Macrophage media (previously prepared) to the pellet: 3ml per mouse.
   • Re-suspend cells by pipetting up and down. At first, do so close to the cell pellet, releasing fluid horizontally over the walls of the tube so as not to lyse the cells. Later, keeping doing this but higher over the cell pellet. This must be done quickly and firmly so as no suspend cells properly, but not too forcefully so that you do not lyse the cells.

Plating Counts: Determining cell concentration in macrophage cell suspensions

1. Prepare dilutions
   • Add 20μl of cell suspension to 80μl of cold PBS (5X dilution).
   • Take 10μl of this: dilution + 10μl of dye (2X dilution).
   • Use 10μl of this 10X dilution and pipette into counting chamber.
2. Counting
   • Count number of cells in each square of the chamber.
   • Average the number for all 4 chambers.
   • Use this number to determine: [Macrophages] = # Average of cells x 10 (DF) x 1x10⁴ = ____ cells/ml
3. Dilute cell suspension
   • You will dilute cell suspension to obtain a concentration of 500000 cells/0.7ml, which will give 500000 cells per well when 0.7ml of this
     suspension is plated. Each of the small wells on slide will contain 0.7ml of cell suspension
   • Determine M₁ by counting procedure under microscope: M₁
   • Determine V₁, this is the actual amount of suspension you have in the tube: V₁ = x
   • Perform calculations as specified bellow, for each mouse.
   • Add the amount of diluent of macrophage media to achieve the desired concentrations: V₂ - V₁ = diluent = y
   • This will give you a cell suspension with your desired cell concentration.

WT	HET KO
M1 = ____ cells/ml M2 = 6x105 cells/0.7ml = 8.57x105 cells/ml V1 = ____ ml V2 = ____ ml DF = M1/M2 = M1/(8.57x105 cells/ml) = V2/V1 = (x+y)/x M1/(8.57x105 cells/ml) = (x+y)/x y = (x * M1)/(8.57x105 cells/ml) - x	M1 = ____ cells/ml M2 = 6x105 cells/0.7ml = 8.57x105 cells/ml V1 = ____ ml V2 = ____ ml DF = M1/M2 = M1/(8.57x105 cells/ml) = V2/V1 = (x+y)/x M1/(8.57x105 cells/ml) = (x+y)/x y = (x * M1)/(8.57x105 cells/ml) - x

Plating

1. Plate 700μl per well
   • You will add 0.7ml of the cell suspension at 600,000 cells/0.7ml into each well of the 8-well slide until all cell suspension has been plated.
   • Add to each well by going against the wall of the plate without actually touching it with pipette.
2. Incubate at 37°C and 5% CO₂ overnight

oxLDL Stimulation

1. Prepare Media
   • Cells will be stimulated in 10% FBS DMEM (high glucose) + Pen-Strep
   • Stimulate cells using 0.7ml/well
   • Cells will be stimulated with following conditions, in triplicate, for each genotype:
     • Media
     • oxLDL 50μM
     • oxLDL 100μM
     • oxLDL 150μM
   • Use M₁V₁ = M₂V₂; V₁ = (M₂V₂)/M₁
2. Wash Cells
   • Add 1X PBS at Room Temperature. Make sure to do this slowly, against the wall of the well so as not to hurt cells.
   • Aspirate PBS with sterile glass Pasteur pipet.
   • Do one slide at a time so that cells are not out of 37°C for too long.
   • Do not let wells dry; add PBS immediately after removing media.
3. Stimulate cells
   • Add 0.7ml/well of appropriate media
   • Stimulate cells for 48 hours

oxLDL (write down information): Amount, Date prepared, Concentration (mg/ml).
Stimulation: Start, End.

Oil Red O Staining
MATERIALS

• 1X PBS - Sterile, does not need to be tissue-culture tested
• 4% Paraformaldehyde:
  • Paraformaldehyde powder (Sigma Cat. No. P-6148) - 0.04267g/ml in PBS, prepare at least 60ml, 2.56g in 60ml 1X PBS.
  • Combine in a clean beaker and stir on stirring plate. While stirring, set on pH meter and add NaOH to dissolve. It will not dissolve right away, but you can add up to a pH~11; then, stop adding NaOH and continue stirring at RT for a few minutes. It takes a few minutes for this to happen. Once dissolved, bring down pH to pH~7 on pH meter.
  • Cool down to 4°C.
• 60% Isopropanol, prepared from 100% Isopropanol (in Flammable cabinets, in a big jar)
• Oil Red O: 0.4% Oil Red O in 100% Isopropanol
• Hematoxylin (Biomeda Cat. No. M10)
• Saturated Sodium Bicarbonate
• Mounting Media (Glycerol Mount, Sigma Cat. No. GG1-15ML)
• ddH₂O

Protocol

1. Wash Cells: 2X with 1xPBS
   • Remove media by carefully aspirating. Do not touch cells. (leave the chambers on!)
   • Add PBS to side of chamber, using cut tips (stream from cut tips will be less forceful)
2. Fix Cells: 10min 4% Paraformaldehyde
   • Prepare 4% Paraformaldehyde fresh, using powder form as follows:
   • 2.56g/60ml (M = 42.67mg/ml or 0.04267g/ml)
   • Heat solution to 65°C until dissolved, shaking repeatedly, then cool to 4°C (good for a couple of days only)
3. Rinse: 60% Isopropanol
   • Rinse quickly in 60% isopropanol, since the lipid is soluble in 100%Isopropanol
   • Make from 100% as follows
   • V₁ = (M₂V₂)/M₁ = (60% x ____ ml)/100% = ____ ml
     
     Ex: 60ml 100% Isopropanol + 40ml H₂O, V₂ = 100ml
4. Oil Red O Lipid Staining: 10 min
   • Oil Red O is kept as 0.4% Oil Red in 100% Isopropanol; Working solution should be diluted to Oil Red in 60% Isopropanol
   • V₁ = (M₂V₂)/M₁ = (60% x 60 ml)/100% = 36 ml
   • 36ml Stock Oil Red + 24ml H₂O
   • Filter stock 1x. After diluted, filter again 1x
5. Rinse
   • 60% Isopropanol quickly
   • Then ddH₂O
6. Counterstain Hematoxylin
   • Filter Hematoxylin
   • Wash first with ddH₂O
   • Add Hematoxylin ~ 4 minutes (a couple of drops)
   • Wash with ddH₂O several times: dip slides in water, change water a few times
   • Add Saturated Sodium Bicarbonate ~ 1 minute (dip in the solution)
   • Rinse with ddH₂O (dip in water)
7. Mounting: Glycerol Mount
   • Let slides dry at RT, then place on hot bed (on Xiuping’s bench) at ~40°C to warm
   • Warm mounting media to 55°C on a beaker over a hot plate; media should become liquid. Keep mounting media there until ready to us
   • Invert mounting media to let bubbles go up, take first drop in Kim wipe
   • Add one drop of Glycerol mount to each section. Carefully, not to touch sections, and without bubbles. If the slide and the mounting media are warm as they should be, the media will spread out on its own
   • To place coverslip, place edge of it on right side of slide and push down slowly with forceps. Go slowly, pushing down toward left edge of slide, this will allow bubbles to get out
   • Make sure all sections are covered with mounting media, push out any bubbles remaining
   • Dry overnight, not tilting slide or mounting media may drip