2023 Fall Stephens

2023 Fall Stephens kdorfman Wed, 07/19/2023 - 14:34
day date topic prep
Tu 9/5 Learn your microscope 10 HeLa parentals, FITC tubulin, 568 phalloidin, DAPI
Th 9/7 Transmitted light and alignment
Tu 9/12 Cameras and calibration
Th 9/14 Numerical Aperture and resolution
Tu 9/19 Fluorescence
Th 9/21 Fluorescence photobleaching (10 brand new triple stained HeLa slides?) Drew says no
Tu 9/26 Immunofluorescence procedure 20 coverslips of fixed HCT116 WT cells (grown on McCoy's) (maybe half treated with a drug the day before fixation),
laminA, green secondary
alpha tubulin, red secondary
Th 9/28 Immunofluorescence imaging
Tu 10/3 Live cell imaging and cell culture Split & seed another dish, live cell staining & imaging nuc blue, mitotracker red
Th 10/5 Plasmid prep. and cell culture Eb1 in liquid culture, Zymo miniprep
Tu 10/10 Monday schedule (Kate transfects HCT116's with Eb1 plasmids, seeds on viewing dishes)
Th 10/12 Transfection imaging / Makeup day image kate-transfected cells
Tu 10/17 Midterm Mai does trial runs
Th 10/19 FLEXIBLE DAY / CURE intro
Tu 10/24 1st day CURE Students fix & stain coverslips
Th 10/26
Tu 10/30
Th 11/1
Tu 11/6
Th 11/8
Tu 11/13
Th 11/15
Tu 11/20
Th 11/22 THANKSGIVING
Tu 11/28
Th 11/30 Final Project due
Tu 12/5 Neurobiology
Th 12/7 Neurobiology

09/26/23 Immuno procedure

09/26/23 Immuno procedure kdorfman Thu, 09/21/2023 - 21:19

Immunofluorescence Procedure Tu 9/26/23

Materials

To mix primaries:

  • Lamin A:
    • Use 4 aliquots of 5 µL each
    • Add 125 µL PBS-Tw-Az-BSA to get (slightly less than) 2X strength
    • total volume: 520 µL
  • Alpha Tubulin:
    • Use 3 aliquots of 4 µL each
    • Add 200 µL PBS-Tw-Az-BSA to get 2X strength
    • total volume = 600 µL
  • Mix 1:1, and aliquot ~104 µL per pair. Make 10 aliquots

To mix secondaries

  • Green anti-mouse:
    • 2 1µL aliquot
    • 300 µL PBS-Tw-Az-BSA
    • total volume: 602 µL
  • Red anti-rat:
    • 2 5 µL aliquots
    • 260 µL PBS-Tw-Az-BSA each
    • total volume: 530 µL
  • Mix 1:1, and aliquot ~106 µL per pair. Make 10 aliquots

10/03/23

10/03/23 kdorfman Mon, 10/02/2023 - 20:54

Live Cell Staining & Imaging

Students use the dish they seeded previous Thursday.

Need "staining solution":

To make staining solution for coverslip bottom dishes
with mL staining solution per dish
Start with: mL Fluorobrite
add: µL Nuc Blue
and µL Mitotracker Red

10/10/23

10/10/23 kdorfman Mon, 10/02/2023 - 19:38

Kate transfects HCT116 cells with Eb1 plasmids.

Need 8-10 viewing dishes, seeded with transfected cells 1 to 2 days before viewing.

1 reaction needs ~ 106 cells/mL

100 uL cells per reaction (barely 8 drops)

So do 2 reactions, each with 106 cells.

2 days before, seed a 25 cm2 flask with 0.5 106cells.

(4 days before, seed with 0.125 106 cells)

1 drop of the 100 uL reaction into its own dish.

On transfection day, divide into 2 reactions

Notes on actual protocol followed:

Friday 10/6

  • Start with 2 confluent 12.5 cm2 flasks
    • (~1.25 x 106 cells)
    • Need 1-5 x 106 cells/mL on Monday
    • Split 1/8 to allow for 3 doublings
    • Stop reaction with 1.6 mL
    • Seed 0.2 mL into each of 2 new flasks.

Monday 10/9/23

  • Put all the cells from one confluent 12.5 cm2 flask
    • should be confluent again
  • into a new 25 cm2 flask

Tuesday 10/10/23

  • There should be 2.5 x 106 cells
  • Trypsinize
  • Stop reaction with 2 mL
  • split suspended cells into two microfuge tubes.
  • Spin down, and proceed with 2 Mirus nucleofection reactions
  • Make 2 12.5 cm2 flasks

10/17/23

10/17/23 kdorfman Mon, 10/16/2023 - 19:26

Trial run for Lamin antibodies

Fix 15 min in paraglut

1o Ab: Rabbit anti-lamin B

  • dilute 1:1000 with PBS-Tw-Az-BSA
  • 2 µL antibody into 2 mL buffer
  • 1 hr 37C

2o Ab: goat anti-rabbit

  • 1 hr RT incubation
Cell type # coverslips
MEF WT NLS-GFP (40) 3
HCT-116 WT (60) 3
DU-145 (74) 4

Stain with Hoechst (1:10,000) in the final PBS-Tw-Az rinse

Mount with non-DAPI Fluoromount (Fisher OB100-01)

10/24/23

10/24/23 kdorfman Mon, 10/16/2023 - 19:36

Students begin projects

Kate provides fixed coverslips (seeded 10/22, fixed 10/24/23)

Cell line medium variant treatment no. coverslips
HCT116 McCoy WT (60) 4
LB1-AID-GFP (68) 2
MEF DMEM WT (39) control 6
VPA
DZNP
LMNB1-/- (02) 3
DMEM+G418 LA KD (37) 3
Prostate cancer RPMI-1640 DU145 (74) 2 +3 from Mai
RPMI-1640 LNCaP (77) 2 + 3 from Mai
DMEM PC3 (76) 2

Materials:

Project people scope IF coverslips IF details
MEF VPA Olivia & Juliana 1 1x 39 unt, 1X 39 VPA, another VPA if pos standard NP LB1 IF
MEF DZNep Karan & Nate 2 1x 39 unt, 1X 39 DZNep, another if pos standard NP LB1 IF
MEF LMNB1-/- Cole & Grif 3 1X 39, 2X 02 LMNB1-/- 39 & 02 NP LB2 IF, 1x 02 NP LB1 IF
MEF LA KD Shrushti, Emma, Anna 8 1X 39, 2X 39 standard NP LB1 IF
HCT116 LB1 live 1 Sam & Jillian 5 68, 1x unt & 1x VPA LB2 IF only
HCT116 LB1 live 2 Isabelle & Jason 6 60, 1x unt 1x VPA; 60, 1x unt 1x VPA one set NP LB1 IF; other set NP LB2 IF
Prostate Cancer 1 Tyian & Antonia 4 2x 74, 1x 76 standard NP LB1 IF
Prostate Cancer 2 Kelsey & Pedro 7 2x 74, 1x 77 standard NP LB1 IF