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Overview Seeding into T- 75 Seeding into 6 well plates Complete media 12hrs Serum starvation /Hypoxia vs replete Cell preparation for flow cytometry 24hrs.

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Presentation on theme: "Overview Seeding into T- 75 Seeding into 6 well plates Complete media 12hrs Serum starvation /Hypoxia vs replete Cell preparation for flow cytometry 24hrs."— Presentation transcript:

1 Overview Seeding into T- 75 Seeding into 6 well plates Complete media 12hrs Serum starvation /Hypoxia vs replete Cell preparation for flow cytometry 24hrs

2 OVERVIEW Seeding into T-75 12 hrs Seeding into 6 well plates Complete media 24 hrs Serum starvation / Hypoxia vs replete Cell preparation for flow cytometry

3 Serum Starvation Only CultureHTR-8/Svneo Trophoblast cells to 80% confluence on 6- well plates Media: RPMI 1640 + Glucose +L-glutamine + Fetal bovine serum (FBS) 10% +Penicillin/strep 100x 12hrs Decant supernatant Wash with 2.0mls DPBS+ Ca+,Mg2+, Add 2.5mls of RPMI1640X 1 (+/-FBS)Place in 5% Co2 incubator 24hrs Prepare for Flow cytometry 2-3hrs

4 Serum Starvation Only CultureHTR-8/Svneo Trophoblast cells to 80% confluence on 6- well plates Media: RPMI 1640 + Glucose +L-glutamine + Fetal bovine serum (FBS) 10% +Penicillin/strep 100x 12hrs Decant supernatant Wash with 2.0mls DPBS+ Ca+,Mg2+, Add 2.5mls of RPMI1640X 1 (+/-FBS) Place in 5% Co2 incubator 24hrs Prepare for Flow cytometry 2-3hrs

5 Serum Starvation Only 12hrs24hrs2-3hrs CultureHTR-8/Svneo Trophoblast cells to 80% confluence on 6- well plates Media: RPMI 1640 + Glucose +L-glutamine + Fetal bovine serum (FBS) 10% +Penicillin/strep 100x Decant supernatant Wash with 2.0mls DPBS+ Ca+,Mg2+, Add 2.5mls of RPMI1640X 1 (+/-FBS) Place in 5% Co2 incubator Prepare for Flow cytometry

6 Serum Starvation+ HR+ RLN/VEH (concurrent) CultureHTR-8/Svneo Trophoblast cells to 80% confluence on 6- well plates Media: RPMI 1640 + Glucose +L-glutamine + Fetal bovine serum (FBS) 10% +Penicillin/strep 100x Decant supernatant Wash with 2.0mls DPBS+ Ca+,Mg2+, Add 2.5mls of RLN100/10/1ng OR VEH 100/10/1ng in RPMI1640X1(-FBS) OR complete medium Place in Hypoxic (2%O2/humidifier) 6hrs 12hrs Remove from Hypoxic chamber Place in 5%Co2 18hrs Flow cytometry 2hrs

7 Serum Starvation+ HR+ RLN/VEH (concurrent) CultureHTR-8/Svneo Trophoblast cells to 80% confluence on 6- well plates Media: RPMI 1640 + Glucose +L-glutamine + Fetal bovine serum (FBS) 10% +Penicillin/strep 100x Decant supernatant Wash with 2.0mls DPBS+ Ca+,Mg2+, Add 2.5mls of RLN100/10/1ng OR VEH 100/10/1ng in RPMI1640X1(-FBS) OR complete medium Place in Hypoxic (2%O2/humidifier) 6hrs 12hrs Remove from Hypoxic chamber Place in 5%Co2 18hrs Flow cytometry 2hrs

8 Serum Starvation+ HR+ RLN/VEH (concurrent) 12hrs6hrs8hrs2hrs CultureHTR- 8/Svneo Trophoblast cells to 80% confluence on 6- well plates Media: RPMI 1640 + Glucose +L-glutamine + Fetal bovine serum (FBS) 10% +Penicillin/strep 100x Decant supernatant Wash with 2.0mls DPBS+ Ca+,Mg2+, Add 2.5mls of RLN100/10/1ng OR VEH 100/10/1ng in RPMI1640X1(-FBS) OR complete medium Place in Hypoxic (2%O2/humidifier) Remove from Hypoxic chamber Place in 5%Co2 Flow cytometry

9 Serum Starvation+ HR+ RLN/VEH (Pre- Treatment) Cell culture: HTR-8/Svneo Trophoblast cells to 80% confluence (12hrs ) Pretreatment: Wash with DPBS x1 Pretreat with Relaxin 100/10/1ng respectively Discard supernatant after 6hrs Add RLN/VEH vs complete media Place in hypoxic (2% O2 /humidifier) x 6hrs Remove from Humidifier and hypoxia Reperfusion /Reoxygenat ion Place in 5% Co2 x 18hrs Remove from incubator Flow cytometry

10 Serum Starvation+ HR+ RLN/VEH (Pre-Treatment) Cell culture: HTR-8/Svneo Trophoblast cells to 80%confluence (12hrs ) Pretreatment: Wash with DPBS x1 Pretreat with Relaxin 100/10/1ng respectively Discard supernatant after 6hrs Add RLN/VEH vs complete media Place in hypoxic (2% O2 /humidifier) x 6hrs Remove from Humidifier and hypoxia Reperfusion / Reoxygenation Place in 5% Co2 x 18hrs Remove from incubator Flow Cytometry

11 Investigating The Mechanism For RLN Rescue Cell culture: HTR-/Svneo Trophoblast cells to 80%conflue nce (12hrs) Pre-Treatment: Wash with DPBS x1 Pre Treat with LY OR Wortmanin OR AKT inh x (30miins) Place in 5% Co2 incubator Remove from incubator RLN/VEH: Wash with DPBS RLN 10ng/VEH 10ng/ complete medium Hypoxic (2%O2)/ humidifier Remove from hypoxia after 6hrs Reperfusion/Reo xygenation: Place in 5%CO2 incubator x 18 hrs Flow cytom etry

12 INVESTIGATING THE MECHANISM FOR RLN RESCUE Cell culture: HTR-/Svneo Trophoblast cells to 80%confluence (12hrs) Pre-Treatment: Wash with DPBS x1 Pre Treat with LY OR Wortmanin OR AKT inh x (30miins) Place in 5% Co2 incubator Remove from incubator RLN/VEH: Wash with DPBS RLN 10ng/VEH 10ng/ complete medium Hypoxic (2%O2)/ humidifier Remove from hypoxia after 6hrs Reperfusion/ Reoxygenat ion: Place in 5%CO2 incubator x 18 hrs Flow Cytometry

13

14 ER stress ( P-ell-2u,CHOP, XBP-1 spliced variant ) Ischacmia/Reperfusion (Selectively) Protein Translation ROS Trolox Edaravone Apoptosis Akt expression Overexpression of Akt Phospho-Akt ( P-BAD (Ser 136) ) LY294002

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