Flow Cytometry Methods.

 

BrdU Staining Protocol with DNase

Supplies:
Ethanol 100% USP (highest quality)
FACS Staining Buffer (1XPBS w/ 3% calf serum, 0.05% azide--filtered) —Dilute staining antibodies in Buffer
DNAse (Sigma D-5025, Bovine Pancreas)
RNase (Boehringer, 25 mg bovine pancrease)
Anti-BrdU-FITC (Becton Dickinson or Phoenix Flow)
0.15 M NaCl, 1.5 M NaCl
10% Paraformaldehype (kept as stock in -80°C)
Tween
1M MgCl2
FACS Tubes

Protocol:

Cells in 96 well FACS plate

  1. Block with 24G-2

  2. Surface stain cells as usual
    -Omit fourth channel labeled antibodies on all stains; EtOH destroys APC

  3. Prepare tubes from which to transfer EtOH drop wise (1.2 ml EtOH on ICE)

  4. Resuspend cells from 96 well place with 100 µl 0.15M NaCl (cold)

  5. Transfer to FACS tubes ON ICE. Add 400 µl 0.15M NaCl to each tube

  6. Vortex at 1/3 speed and add EtOH with pasteur pipette at 1 drop per second.
    This is a critical step... do not add EtOH too quickly

  7. Incubate on ice for 30 minutes

  8. Spin 10 minutes @ 2000 RPM, 4° C

  9. Dump and shake liquid into waste

  10. Using repeat pipetter, squirt 1 ml FACS staining buffer into each tube

  11. Spin 10 minutes and dump as before (step 8)

  12. Add 1 ml 1% paraformaldehyde + 0.05% Tween 10
    -For 20 ml:
    2.0 ml 10% paraformaldehyde
    10 µl Tween-20

  13. Incubate at room temperature for 30 minutes

  14. Incubate on ice for 30 minutes

  15. Spin and dump as before (step 8)
    Add 1 ml DNAse (0.15M NaCl + 4.2mM MgCl + 100 Kunitz units/ml DNAse)
    -For 50 ml:

46.5 mL dH20
200 ul MgCl2 (1M stock)
1500 uL NaCl (5M stock)
100 Kunitz units Dnase (volume depends on activity of batch)
Incubate for 30 minutes @ 25°

  1. Spin 10 min. and dump as before (step 8)

  2. Transfer cells from FACS tubes to 96-well plate. Wash once with staining media.

  3. Block with 10% rat serum. Incubate 15 minute on ice. Spin and dump as before (step 8: it is critical to spin at high speed once the cells have been fixed with EtoH/ PFA since they become less dense).

  4. Add anti-BrdU-FITC or biotin (1:20 dilution for Phoenix flow).

  5. Pipette up and down to resuspend pellet. Incubate for 30 minutes on ice (or overnight at 4°C).

  6. Wash and dump as before. Transfer cells into FACS tubes.

BrdU staining for cell cycle analysis (with HCl treatment)

A total of 3x106 retinoblastoma cells were treated with cytokines, at the concentration noted above, for 72 h in medium with 10% FBS. The cells were then pulsed with 20 μm BrdU (Sigma, St. Louis, MO) for one hour at 37 °C in 10% FBS. The cells were then fixed with cold 95% ethanol, washed with PBS and resuspended in 0.4 mg/ml pepsin in 0.1 N HCl for 30 min at room temperature (RT) to release nuclei. The nuclei were pelleted and incubated with 2 N HCl for 30 min at RT. Following neutralization with 0.1 M Na2B4O7, nuclei were pelleted and washed with PBS + 0.5% Tween-20 + 0.1% BSA. Nuclei were then stained with anti-BrdU antibody (Becton-Dickinson, Franklin Lakes, NJ) for 90 min in the dark at RT. The nuclei were then washed with PBS, pelleted and stained with FITC-labeled goat anti-mouse antibody (1:50, Becton-Dickinson) for 30 min at RT in the dark. The nuclei were washed with PBS, incubated with propidium iodide (0.1 mg/ml) and RNAse A (10 μg/ml) overnight at 4 °C, and then analyzed using a FACScalibur machine. Because of extensive aneuploidy, we chose to quantitate only the non-aneuploid portions of the FACS. The portions of the FACS analysis used for gating each population of cells were between 200 and 400 on the X-axis. The percentages, therefore, do not total 100%. However, the percentages total to over 70% for all samples thus represent the majority of cells in the population

 

FACS Analysis of Endothelial Cells
(Karin Ebner)

.)cell starving: 24h with serumreduced Medium (5% serum)

.)Incubation with e.g. TNF (24h)

  for max. stimulation of ICAM-1 we added IL-1ß (20 Units/ml) for the last 24h hours of

  incubation

.)after incubation wash cells with cold PBS, scrap the cells from the plate in

  PBS+5%Serum into a tube and put them on ice (no trypsin)

.)centrifugation: 4°C/1.500g/15min 

.)cell pellet + first antibody -we incubated the cells for 15 min on ice and shaked the

                                cells during this 15 min a few times (not vortexing)

.)stop the reaction with 1ml PBS+5%Serum, centrifugation: 4°C/1.500g/5min

.)wash cells once in PBS+5%Serum

.)cell pellet + second antibody -we incubated 30 min on ice (sometimes shaking the cells)

.)stop the reaction and wash cells as above, afterwards resuspend the cells in PBS for

  FACS-analysis

 

The use of PBS with 5% Serum may sound strange. We used this for a good treatment of the sensitive primary endothelial cells. Could be that this is not necessary.

There is also a publication which might be interesting:

KIM JA, Atherioscler Thromb 14: 427-433, 1994

 

JC-1 stain of apoptotic cells

JC-1 (5, 5´, 6, 6´-tetrachloro-1, 1´, 3, 3´-tetraethylbenzimidazol-carbocyanine iodide) is a lipophilic fluorescent cation that incorporates into the mitochondrial membrane, where it can form aggregates due to the physiological membrane potential of mitochondria. This aggregation changes the fluorescence properties of JC-1 leading to a shift from green to orange fluorescence.

Intact living cells stained with JC-1 therefore exhibit a pronounced orange fluorescence of mitochondria, which is detectable by flow analysis (in the FL-2 channel).

Apoptosis results in a break-down of the mitochondrial membrane potential and a subsequent decrease of the orange fluorescence (and a slight increase of the green fluorescence). By that means, apoptotic cells can be easily distinguished from non-apoptotic cells.

JC-1 is prepared as a 1000x stock solution in DMSO (5 mg/ml).

For the staining of adherent cells it is diluted in medium to 5 µg/ml (with vortexing during the dilution to prevent the formation of precipitates); the JC-1 containing medium is added to the cells, followed by incubation for 10 min at 37°C (or RT for 15 min).

Subsequently the cells are washed twice with PBS, trypsinized, suspended in 500 µl PBS and analyzed by flow analysis.

Suspension cells (lymphocytes): suspend 1:1 with 10 µg/ml JC-1 in medium (final conc.: 5 µg/ml)

 

Approximate detection settings on FACSort:

FL1: 360 V (log)

FL2: 310 V  (log)

Compensation : FL1-7% FL2 und FL2-74% FL1

Andere Einstellungen: Zelltyp-spezifisch, bei TF-1 z.B.:

SSC: 336 V lin

FSC: E00 lin 1.0

Threshold: FSC: 52

 

Example of an analysis:

(modified from Andrea Cosarizza)

 

Cell Cycle Analysis by Propidium Iodide (PI) Staining

Adherent cells:

  • trypsinized

  • suspended in medium + 10% FCS

  • centrifuged (1000 rpm, 5 min)

  • Pellet suspended in PBS (1 ml)

 

Suspension cells:

  • Centrifuged (1000 rpm, 5 min)

  • Pellet suspended in PBS (1 ml)

 

Fixation with EtOH:

Pipet cell suspension into 2.5 ml absolute EtOH (final concentration approx. 70%)

(or vortex the suspension at half speed while adding the EtOH) – to prevent clustering of cells during the fixation. Incubate on ice for 15 min (or over night at –20°C).

 

Alternative fixation with paraformaldehyde:

Pipet the 1 ml cell suspension into 3 ml 4% paraformaldehyde and fix for 15 min at r.t.

 

Staining:

  • Pellet the cells at 1500 rpm for 5 min

  • Suspend the pellet in 500 µl PI-solution
    in PBS: 50 µg/ml PI from 50x stock solution (2.5 mg/ml)
    0.1 mg/ml RNase A
    0.05% Tritin X-100
    Incubate for 40 min at 37°C

  • Add 3 ml of PBS, pellet the cells (1500 rpm, 5 min) and take off the supernatant

  • Suspend the pellet in 500 µl PBS for flow analysis
    (you can also leave about 500 µl of the diluted staining solution on the pellet and suspend the cells in this solution > less loss of cells when you take off the sup.) – the rest of the staining solution does not interfere with the flow analysis.

 

Flow analysis:

Approximate settings (on FACSort):

FL1: 570 V log. (e.g. if you want to detect GFP):

FL2: 470 V linear  

 

Example: