AUTHOR: Tim Starr                Date last revised: 12/29/03

 

 

Standard Operating Procedure

 

 

Intracellular staining for Flow Cytometry or Microscopy

 

 

Hazards:

 

* Formaldehyde, paraformaldehyde, sodium azide and 100% methanol are harmful.

 

Protection:

 

* Wear gloves when handling formaldehyde, paraformaldehyde or any buffers containing sodium azide.

 

Waste:

 

* Dispose of formaldehyde, paraformaldehyde and methanol using standard hazardous waste disposal method.

 

Spill Clean-Up:

 

* Follow normal procedures.

 

Procedure:

 

Overview: Tagging intracellular molecules with fluorescent antibodies requires fixation and permeabilization. Different cell types and molecules will require different fixation and permeabilization chemicals at varying concentrations. You may need to try various fixatives and permeabilizing agents at different concentrations, times and temperatures before finding the optimal procedure. You can also stain for extracellular molecules at the same time as you stain for intracellular molecules. However, the fixatives and perm agents may damage the epitope required for staining. If this occurs, you can try staining for the extracellular molecules before fixing or before permeabilization. Listed below are four procedures that have worked in our hands for the indicated molecules and cells.

 

Paraformaldehyde and Saponin method #1: For PKC theta

Paraformaldehyde and Saponin method #2: For IL-2

Formaldehyde & methanol method: For phospho-ERK

Formaldehyde & saponin method: For phospho-MAPk and cJun (Rosette protocol)

 

 

Paraformaldehyde and Saponin method #1:

 

Works for staining PKCtheta in thymocytes and T cells

 

Generally done in 96 well-plates with 1e6 cells/well. Spins are done at 4ºC for 5 min at 1000 rpm (150 G).

 

 

·      Fix cells: Resuspend pelleted cells in 200 µl 1% PFA. Let sit RT 20 min. Spin & Flick.

 

·      Wash cells: Resuspend in 200 µl Perm buffer. Spin & flick.

 

·      Intracellular 1º stain: Stain with 1º antibody (eg. Anti-PKCq @ 1:100) and a sample with an isotype or normal sera in 100 µl perm buffer for 30 min on ice (note: I have also done this 60-90 min RT).

 

·      Wash cells: Resuspend in 200 µl Perm buffer. Spin & flick.

 

·      Intracellular 2º stain: Stain with 2º antibody (eg. Bio-Goat anti-Rabbit @ 1:100) in 100 µl perm buffer for 30 min on ice.

 

·      Wash cells: Resuspend in 200 µl Perm buffer. Spin & flick.

 

·      Intracellular 3º stain: Stain with 3º antibody (eg. SA-Alexa 568 @ 1:100) in 100 µl perm buffer for 30 min RT.

 

·      Wash cells : Resuspend in 200 µl Perm buffer. Spin & flick.

 

·      Wash cells: Resuspend in 200 µl Stain buffer. Spin & flick

 

·      Extracellular 4º stain: Stain with extracellular antibody (eg. FITC-CD45 @ 1:100) in 100 µl stain buffer for 30 min on ice.

 

·      Wash cells 2 x: Resuspend in 200 µl Stain buffer. Spin & flick. Repeat

 

·      For FACS analysis: Resuspend pellet in 200 µl stain buffer and analyze by FACS.

 

·      For microscope analysis: Do not resuspend. Pipet 20 µl of what remains after flicking in 96 well plate onto microscope slide. Add one drop of Mowiol/DABCO mounting/anti-fade reagent. Cover with coverslip. Put two drops of nail polish on corners of slide to hold in place. Let dry overnight at room temp. Store at 4ºC. View in microscope.

 

Stain Buffer = 2% FCS, 0.2% NaN₃, in PBS.

Perm Buffer = Stain Buffer + 0.1% saponin. (note – can play with % saponin if you want).

 

 

Paraformaldehyde and Saponin method #2:

 

Works for staining IL-2 in thymocytes and T cells

 

Generally done in 96 well-plates with 1e6 cells/well. Spins are done at 4ºC for 5 min at 1000 rpm (150 G).

 

 

 

·      Fix cells: Resuspend pelleted cells in 200 µl 2% PFA in PBS. Let sit RT 20 min. Spin & Flick.

 

·      Wash cells: Resuspend in 200 µl PBS. Spin & flick.

 

·      Stain extracellular: Resuspend in 100 µl stain buffer + extracellular Abs. Let sit on ice 30 minutes. Spin & Flick.

 

·      Wash cells: Resuspend in 200 µl Perm buffer. Spin & Flick.

 

·      Wash cells: Resuspend in 200 µl SuperPerm buffer, let sit 10 minutes RT. Spin & Flick

 

·      Stain: Resuspend in 100 µl Perm buffer + Ab. Let sit 30 min RT. Normally use antibody at ?µg/ml

 

·      Wash cells: Resuspend in 200 µl Perm Buffer. Spin & Flick.

 

·      Wash cells: Resuspend in 200 µl PBS. Spin & Flick

 

·      Wash cells: Resuspend in 200 µl Stain buffer. Spin & Flick

 

·      FACS: Resuspend in 200 µl and FACS analyze.

 

 

Stain Buffer = 2% FCS, 0.2% NaN₃, in PBS (pH 7.2 – 7.4)

Perm Buffer = Stain Buffer + 0.5% saponin

SuperPerm Buffer = 3 parts Perm buffer + 1 part filtered FCS

 

Formaldehyde & methanol method:

 

Works for staining phospho-ERK in thymocytes and T cells

 

Generally done in 96 well-plates with 1e6 cells/well or in FACs tubes. Spins are done at 4ºC for 5 min at 1000 rpm (150 G). Note: If you are using small quantities of cells, avoid using 1.5 ml eppendorf tubes as you will lose most of your cells following this procedure. Instead, use the polystyrene FACs tubes until you can transfer to 96 well plates.

 

·      Fix: After stimulating cells (note: phospho-ERK occurs rapidly and will extinguish rapidly) either spin & flick and or add enough concentrated formaldehyde (use 10% ultrapure EM grade formaldehyde stock from Polysciences Cat # 04018) to dilute to 2% and incubate in 2% formaldehyde/PBS for 10 – 15 min RT. Note: do not wash at this point. Spin & flick and proceed to next step.

 

·      Perm: Resuspend in ice cold methanol (100%). Incubate 15 - 30 minutes on ice. Spin & wash in wash buffer (200 µl for 96 well plates or 1 ml for FACS tubes) spin & remove supernatant

 

·      Block: Resuspend in 10% 2.4G2 supernatant, 1% normal mouse sera, 1% normal rat sera, and 0.2% NaN₃ in PBS for 10 min room temp. Spin & wash in 200 µl wash buffer.

 

·      Stain cells: Incubate with primary Ab 15 min room temp. Wash one time in wash buffer. Repeat for secondary, tertiary antibodies. Can add cell surface antibodies on the last staining.

 

·      Currently using Cell signaling anti—phospho-ERK (9106) at 1:200, bio-anti-IgG1 (1:500) and SA – PE (1:200) . Using pharmingen isotype anti-MOPC IgG1.

 

·      Analyze by FACs. Have not looked using the microscope.

 

Wash buffer = 4% FCS in PBS.

 

Formaldehyde & saponin method:

 

Works for staining phospho-MAPk & phospho-cJun (according to Rosette) in thymocytes and T cells

 

Generally done in 96 well-plates with 1e6 cells/well or in FACs tubes. Spins are done at 4ºC for 5 min at 1000 rpm (150 G).

 

·      Fix cells: Resuspend pelleted cells in 4% formaldehyde 15 min RT.

 

·      Block Fc receptors: Resuspend in 10% 2.4G2 supernatant, 1% normal mouse sera, 1% normal rat sera, and 0.2% NaN₃ in PBS for 20 min on ice.

 

·      Stain for extracellular markers in 100 µl stain buffer plus desired antibodies for 30 min on ice. Wash twice with stain buffer

 

·      Permeabilize cells: Resuspend in perm buffer for 5 min on ice. Spin & flick.

 

·      Stain cells: Incubate with antibody in 100 µl perm buffer plus antibody 30 min RT. Wash 3 times in perm buffer. Repeat for 2º and 3º antibodies. Final wash in stain buffer.

 

·      Analyze by FACs. Have not looked using the microscope.

 

Stain buffer = 2% FCS, 3 mM sodium azide in PBS.

Perm buffer = 0.1% saponin, 10% FCS in PBS