materials and methods :
DESCRIPTION
Supplementary Material 3 : Qualities of the antisera directed against Hsmar1, SET, THAP4 or Charlie for antibody staining on HeLa cells transiently expressing or not Hsmar1, SETMAR, THAP4 or CHARLIE8. . - PowerPoint PPT PresentationTRANSCRIPT
Reliability of the nanopheres-DNA immunization technology to produce polyclonal antibodies directed against human neogenic proteins
Ahmed Arnaoty, Valérie Gouilleux, Sophie Casteret, Bruno Pitard,
Yves Bigot and Thierry Lecomte
Materials and Methods: Cells were plated at a density of 5x104 cells per well in 1 cm2 Lab-TekTM chamber slides (Fisher Scientific, Waltham, MA, USA) and grown in DMEM (Gibco/Life Technologies, Paisley, UK) supplemented with 10% heat-inactivated fetal bovine serum (PAA Laboratories, Pasching, Austria) at 37°C in a humidified atmosphere containing 5% CO2 for 24 h to 80% confluence. Transfections were performed as described in the main manuscript using 500 ng of pVAX plasmid and jetPEI™ (Polyplus Transfection, Illkirch, France) at an N/P ratio of 5 in Opti-MEM (Invitrogen/Life Technologies) following the Manufacturer’s instructions. The day after transfection, cells were washed twice with 1X-PBS, fixed with 3.7% paraformaldehyde for 5 min at room temperature (RT), washed three times for 5 min with 1X-PBS at RT, before to be permeabilized in 1X-PBS, 0.2% Triton X100 for 5 min at RT. After 2 washes with 1X-PBS, cells were blocked with 1X-PBS, % bovine serum albumin (BSA) for 30 min at RT. Cells were incubated for 1 h at room temperature with a 1/20, 1/50 or 1/100 of a mouse antiserum (directed against Hsmar1, SET, Charlie 8 and THAP4) in 1X-PBS, and 1% BSA. Cells were then washed three times for 5 min with 1X-PBS before being incubated for 1 h at RT with the secondary antibody (AcII; Alexa Fluor® 488 Goat Anti-Mouse, Invitrogen/Life Technologies). Slides were washed three times for 5 min with 1X-PBS. Nuclei were stained with Hoechst 33342 for 5 min at RT. Slides were mounted with Vectashield mounting medium (Vector Laboratories, Burlingame CA, USA) for epifluorescence microscopicobservation. Slides were analyzed using a Nikon Eclipse Ti and NIS-Elements software.The specificity of the antisera directed against Hsmar1 or SET was monitored using HeLa cells transiently expressing Hsmar1 or SETMAR (SET fused to Hsmar1). Negatives controls were also done for each antiserum to verify the preimmune serum and the quality of the secondary antibody on each type of HeLa (transfected with a plasmid or not).
Supplementary Material 3 : Qualities of the antisera directed against Hsmar1, SET, THAP4 or Charlie for antibody staining on HeLa cells transiently expressing or not Hsmar1, SETMAR, THAP4 or CHARLIE8.
S3.1 Immunofluorescence results Murine Balb c antiserum N°1 directed against Hsmar1 on HeLa transfected by pVAX-Hsmar1
plasmid
Antisera N°1Hela with Hsmar1Hela without Hsmar1
++
++
1/20
1/50
1/100
AntiserumAc II
Preimmune serum (PI)Hela with Hsmar1Hela without Hsmar1
PIAc II
++ +
+
1/20
Negative controlsHela with Hsmar1Hela without Hsmar1
Antiserum
Ac II
-
+
-
+
S3.2 Immunofluorescence results Murine Balb c antiserum N°1 directed against Hsmar1 on HeLa transfected with the pcDNA-
3.1-TOPO-Metnase plasmid
Preimmune serum (PI)HeLa with SETMARHeLa without SETMAR
PIAc II
++ +
+
1/20
Immune serum N°1HeLa with SETMARHeLa without SETMAR
++
++
1/20
1/50
1/100
AntiserumAc II
Negative controlsHeLa with SETMARHeLa without SETMAR
AntiserumAc II
-+
-+
S3.3 Immunofluorescence results Murine Balb c antiserum N°2 directed against Hsmar1 on HeLa transfected by pVAX-Hsmar1
plasmid
Negative controlsHela with Hsmar1Hela without Hsmar1
AntiserumAc II
-+
-+
Antisera N°2Hela with Hsmar1Hela without Hsmar1
++
++
1/20
1/50
1/100
AntiserumAc II
Preimmune serum (PI)Hela with Hsmar1Hela without Hsmar1
PI
Ac II
+
+ +
+
1/20
S3.4 Immunofluorescence results Murine Balb c antiserum N°2 directed
against Hsmar1 on HeLa transfected with the pcDNA-3.1-TOPO-Metnase plasmid
Negative controlsHeLa with SETMARHeLa without SETMAR
AntiserumAc II
-+
-+
Preimmune serum (PI)HeLa with SETMARHeLa without SETMAR
PIAc II
++ +
+
1/20
Antisera N°2HeLa with SETMARHeLa without SETMAR
++
++
1/20
1/50
1/100
AntiserumAc II
S3.5 Immunofluorescence results Murine Swiss antiserum N°1 directed against
SET on HeLa transfected by pVAX-Hsmar1 plasmid
Negative controlsHela with Hsmar1Hela without Hsmar1
AntiserumAc II
-+
-+
Antisera N°1Hela with Hsmar1Hela without Hsmar1
++
++
1/20
1/50
1/100
AntiserumAc II
Preimmune serum (PIHela with Hsmar1Hela without Hsmar1
PIAc II
++ +
+
1/20
S3.6 Immunofluorescence results Murine Swiss antiserum N°1 directed against SET on HeLa transfected with the pcDNA-3.1-
TOPO-Metnase plasmid
Negative controlsHeLa with SETMARHeLa without SETMAR
AntiserumAc II
-+
-+
Preimmune serum (PI)HeLa with SETMARHeLa without SETMAR
PIAc II
++ +
+
1/20
Antisera N°1HeLa with SETMARHeLa without SETMAR
++
++
1/20
1/50
1/100
AntiserumAc II
S3.7 Immunofluorescence results Murine Swiss antiserum N°2 directed against
SET on HeLa transfected by pVAX-Hsmar1 plasmid
Negative controls
Hela-HsMAR1/+Hela-HsMAR1/-
AntiserumAc II
-+
-+
Antisera N°2HeLa-Hsmar1HeLa without Hsmar1
++
++
1/20
1/50
1/100
AntiserumAc II
Preimmune serum (PI)HeLa with Hsmar1HeLa without Hsmar1
PIAc II
++ +
+
1/20
S3.8 Immunofluorescence results Murine Swiss antiserum N°2 directed against SET on HeLa transfected with the pcDNA-3.1-
TOPO-Metnase plasmid
Negative controls
HeLa with SETMARHeLa without SETMAR
AntiserumAc II
-+
-+
Preimmune serum (PI)HeLa with SETMARHeLa without SETMAR
PIAc II
++ +
+
1/20
Immune serum N°2HeLa with SETMARHeLa without SETMAR
++
++
1/20
1/50
1/100
AntiserumAc II
S3.9 Immunofluorescence results Murine Balb c antiserum N°2 directed against
Charlie8 on HeLa transfected by pVAX-Charlie8 plasmid
Negative controlsHeLa with Charlie 8HeLa without Charlie 8
AntiserumAc II
-+
-+
Antisera N°2HeLa with Charlie 8HeLa without Charlie 8
++
++
1/20
1/50
1/100
AntiserumAc II
Preimmune serum (PI)HeLa with Charlie 8HeLa without Charlie 8
PIAc II
++ +
+
1/20
S3.10 Immunofluorescence results Murine Balb c antiserum N°2 directed against
Charlie8 on HeLa transfected by pVAX-Charlie8 plasmid
Antisera N°5HeLa with Charlie 8HeLa without Charlie 8
++
++
1/20
1/50
1/100
AntiserumAc II
Preimmune serum (PI)HeLa with Charlie 8HeLa without Charlie 8
PIAc II
++ +
+
1/20
Negative controlsHeLa with Charlie 8HeLa without Charlie 8
Antiserum
Ac II
-
+
-
+
S3.11 Immunofluorescence results Murine Balb c antiserum N°2 directed
against THAP4 on HeLa transfected by pVAX-THAP4 plasmid
Negative controlsHeLa with THAP4HeLa without THAP4
AntiserumAc II
-+
-+
Antisera N°2HeLa with THAP4HeLa without THAP4
++
++
1/20
1/50
1/100
AntiserumAc II
Preimmune serum (PI)HeLa with THAP4HeLa without THAP4
PIAc II
++ +
+
1/20
S3.12 Immunofluorescence results Murine Balb c antiserum N°4 directed
against THAP4 on HeLa transfected by pVAX-THAP4 plasmid
Negative controlsHeLa with THAP4HeLa without THAP4
AntiserumAc II
-+
-+
Antisera N°4HeLa with THAP4HeLa without THAP4
++
++
1/20
1/50
1/100
AntiserumAc II
Preimmune serum (PI)HeLa with THAP4HeLa without THAP4
PIAc II
++ +
+
1/20