eamag/protocols-with-steps huggingface dataset:
TITLE: Electron microscopy (EM) analysis of LRRK2-Nanotube assembles
AUTHORS: Xinbo Wang, Pietro De Camilli

[DESCRIPTION]
This protocol details methods for the analysis of LRRK2-Nanotube assembles by negative stained EM and Cryo-EM.

[STEPS]
SECTION: Negative stained EM analysis
1. Prepare samples in a PCR tube with 300 nanomolar (nM) LRKK2, 20 micromolar (µM) lipid nanotubes and 1 millimolar (mM) GMPPNP.
SECTION: Negative stained EM analysis
2. Incubate samples at 37 °C for 30 min.
SECTION: Negative stained EM analysis
3. Glow-discharge carbon-coated grids (25 mA, 45 s) during the sample incubation time.
SECTION: Negative stained EM analysis
4. Place the discharged grids on a piece of parafilm.
SECTION: Negative stained EM analysis
5. After incubation, apply 6 µL of the samples to the grid and adsorbed on the grid for 5 min at Room temperature.
SECTION: Negative stained EM analysis
6. Blot the grid with filter paper and stained with 2% uranyl acetate for 40 s.
SECTION: Negative stained EM analysis
7. Dry the grid with filter paper.
SECTION: Negative stained EM analysis
8. Collect images using a Talos L 120C TEM microscope at 80 kV with Velox software and a 4k × 4K Ceta CMOS camera (Thermo Fisher Scientific).
SECTION: Cryo-EM analysis
9. Dialyze freshly purified LRRK2 into the Low salt buffer.
SECTION: Cryo-EM analysis
10. After dialysis, incubate LRRK2 (2 micromolar (µM)) with the kinase inhibitor MLi-2 (5 micromolar (µM)) for 10 min on ice.
SECTION: Cryo-EM analysis
11. Add 20 micromolar (µM) lipid nanotubes into the mixture above and further incubate for 60 min at Room temperature in the presence of 1 millimolar (mM) GTP.
 

SECTION: Cryo-EM analysis
12. Glow-discharge C-flat™ holey carbon gold grids (CF-1.2/1.3-3Au) (15mA, 45 s) during the sample incubation time, then place the discharged grids on a piece of parafilm.
SECTION: Cryo-EM analysis
13. After incubation, apply 4 µL of the samples to the grid.
SECTION: Cryo-EM analysis
14. Plunge-freeze sample-loaded grids in liquid ethane-propane mixture using a Vitrobot Mark IV (FEI) with the following parameters: blot force, 0; blot time, 1 s; wait time, 30 s; drain time,  ; humidity, 100%.
SECTION: Cryo-EM analysis
15. Collect cryo-EM micrographs on a Titan Krios transmission electron microscope (Thermo Fisher Scientific) operating at 300 kV, equipped with a post column GIF quantum energy filter and a Gatan K3 Summit DED camera (Gatan, Pleasanton, CA, USA).
SECTION: Cryo-EM analysis
16. Perform the data collection with the SerialEM software. Record movies in super-resolution mode with a physical pixel size of 1.098 A˚ (super-resolution pixel size is 0.549 A˚) and a defocus range of -  to -  .
TITLE: Sleep scoring using Neuroscore
AUTHORS: daniel.dautan daniel, Per Svenningsson

[DESCRIPTION]
Analysis of sleep data using Neuroscore v.30

[STEPS]
1. Open the file using File – Open Recording – Browse – Open
2. Remove unused signals using the right side tools “signal & data” and unselect all markers: 
 

3. Identify the EEG and EMG signals by adjusting the time windows:
 

4. If the signal is unusable, just move to another recording. Here the top signal is EMG and bottom is EEG. You can see clear changes in EMG corresponding to movement and nice oscillation in EEG, the signal is as expected.
5. You can adjust the vertical scale for each signal using the right+click on the axis and scale to fit.
 

6. Once the signal is identified, you can proceed to sleep scoring. The built-in sleep scoring allows perfect scoring of frontal cortex EEG/EMG.
 

7. In inputs signal, select EEG channel for both Cortical EEG and theta EEG, EMG for EMG and activity for activity. Adjust the vertical scale to confirm the selection of correct signals.
 

16. Copy this to excel:
a) Using the time you can extract the total number of sleep events, number of Awake, REM and NREM and thus express it onto %

 

b) You can extract the % of time in dark/light phase based on the timestamps
c) You can average/stdev EMG signal and thus express is as a z score

 

 
Using the z-score of the EMG, you can detect significant increase (SD >2SD) during REM events. This will be the rem sleep behavior disorders events.

 

 

15. Select the data that need to be analyzed:
- time stamps normally 20s
-sleep scoring S is NREM, P is REM, W is awake and X is artifact
- select add column – select EMG channel – select statistical Root Mean Square

 

14. Extract the data.

 

13. You should have sleep scoring for all your recordings on top of the signals.

 

12. Press score study.

 

11. In advanced, select delta oscillation starting at 0.5Hz.

 

10. Select the stage transition as below:
 

9. In artifact detection, select the threshold based on the signal of the recordings. Move the time windows to avoid cutting off signals.

 

8. In scoring rules, select “score active wake as awake.”
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<div class="column">The steps for preparing the lysate are different depending on the starting material. Please ensure you follow the correct procedure for your starting material (see the section <a href="https://www.protocols.io/view/genomic-dna-removal-and-total-rna-purification-fro-f48bqzw?guidelines" target="_blank">Disrupting and Homogenizing Starting Materials</a> and the different lysate preparation protocols <a href="https://www.protocols.io/view/isolate-ii-biofluids-rna-kit-f5kbq4w" target="_blank">here</a>). The subsequent steps detailed in <a href="https://www.protocols.io/view/genomic-dna-removal-and-total-rna-purification-fro-f48bqzw" target="_blank">Genomic DNA removal and total RNA purification</a> are the same in all cases.</div>
<div class="column"> </div>
<p>Follow this protocol for isolating RNA from free viral particles.</p>

[BEFORE_START]
<div>
<div>
<div>
<div class="b-start-block editable-content">
<div class="page" title="Page 12">
<div class="layoutArea">
<div class="column">
<ul>
<li>
<p>Ensure Lysis Buffer RX is prepared (see guidelines).</p>
</li>
<li>
<p>Ensure that all solutions are at room temperature before use.</p>
</li>
<li>
<p>Two types of spin columns are provided with this kit: the <strong>Genomic DNA Removal Column</strong></p>
<p>(blue ring) and the <strong>RNA Column</strong> (black ring). Ensure the correct column is used for each</p>
<p>step of the procedure.</p>
</li>
<li>
<p>All centrifugation steps are carried out in a benchtop microcentrifuge at 14,000 x g except</p>
<p>where noted. Perform all centrifugation steps at room temperature.</p>
</li>
<li>
<p>It is important to work quickly during this procedure.</p>
</li>
</ul>
</div>
</div>
</div>
</div>
</div>
</div>
</div>

[GUIDELINES]
<p>It is recommended that no more than 100 μL of viral suspension be used in order to prevent clogging of the column.</p>
<p> </p>
<p>Please review the Guidelines under <a href="https://www.protocols.io/view/genomic-dna-removal-and-total-rna-purification-fro-f48bqzw?guidelines" target="_blank">Genomic DNA removal and total RNA purification from all types of lysate</a> for other important details.</p>

[STEPS]
?. 
?. 
?. 
?.
TITLE: Chimeric Protein-LAG and Protein-AG sandwich ELISA  
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This ELISA was used to study the interactions between protein-LAG (PLAG) and protein-AG (SpAG) with different immunoglobulin preparations from mammalian and avian species. </div></div>

[STEPS]
?. This ELISA was used to study the interactions between protein-LAG (PLAG) and protein-AG (SpAG) with different immunoglobulin preparations from mammalian and avian species. The 96 well microtiter plate was coated overnight at 4°C with 2 µg/µl per well of PLAG in carbonate-bicarbonate buffer pH 9.6.
?. The plate was then treated with bovine serum albumin solution and washed 4X with PBS-Tween. 50 µl of immunoglobulins (1 mg/ml) is added and incubated for 1h at room temperature, and the microplate is rewashed 4X with PBS-Tween.
?. Then, 50 µL of peroxidase-labeled SpAG conjugate diluted 1:5000 in PBS-non-fat milk was added to each well and incubated for 1h at RT. The plate was washed 4X with PBS-Tween.
?. Then, 50 µL of o-phenylenediamine solution (4 mg/mL) was added, and the plate was incubated for 15 min at RT in the dark. The reaction was stopped with 50 µL of a 3M H2SO4 solution.
?. The plate was visually assessed for color development and read on a microplate reader at 492 nm. A cut-off point was calculated as the mean of the optical density of the negative controls multiplied by two. The cut-off value was set to 0.36.
TITLE:  Fungal Mounting and Staining Procedure
AUTHORS: Yin-Tse Huang

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Fungal Mounting and Staining Procedure</span></div></div>

[STEPS]
?. [Staining]
Commercial Lugol’s Iodine (5%): to examine amyloid, inamyloid, and dextrinoid reaction of basidios (mostly spores) and ascos (mostly asci). Dilute the commercial (5%): distilled water = 1:4 as working solution. (c.a. 1/6)Pictures from mushroomexpert.com & Wikipedia
?. [Staining]
?. [Mounting]
Permanent mounting media Apply as using water, sealing free
?. [Mounting]
2% KOH 1. Clarifying mounts and making tissues and structures visible. (mushroomexpert.com)2. Is used to test for color changes. KOH is used in the identification of many mushrooms, including boletes, polypores, and gilled mushrooms. For boletes, place a drop of KOH on the cap, stem, sliced flesh, and pore surface. For polypores, apply the KOH to the flesh and the cap surface. For gilled mushrooms, place a drop on the cap surface. Note any color changes that take place. A change to yellow is sometimes found in species of Agaricus and Amanita; magenta or olive reactions can help identify species of Russula and Lactarius; deep red or black reactions can help sort out many gilled mushrooms; black reactions among polypores are crucial separators; and various colors are produced with boletes. Don't forget that a "negative" reaction (no color change) may also be an informative character! (mushroomexpert.com)10% KOH (Dermatology use)As mounting solution to dissolves the background keratin, unmasking the fungal elements to make them more apparent.
?. [Mounting]
Water mountingRegular mounting solution for every kinds of fungal tissue.
TITLE: Optogenetically inhibiting enteric neurons in the murine large intestine
AUTHORS: Dante  Heredia, Thomas  Gould

[DESCRIPTION]
Protocol for optogenetically inhibiting neuronal subtypes in murine colonic myenteric plexus. 

[GUIDELINES]
This protocol applies to transgenic animals expressing the optogenetic protein halorhodopsin in colonic myenteric neurons. 

Although this technique apparently inhibits some populations of cells, we were never able to see any effects  of any amount of light stimulation of cholinergic or nitrergic myenteric neurons on colonic smooth muscle tension.

[STEPS]
1. Dissection and experiments are done in the dark under infrared illumination. A ventral midline incision is made and the whole colon is carefully excised into a Sylguard lined dissection dish containing oxygenated Krebs-ringer solution.
2. The colon is then drawn over a 1.5-mm diameter fire-polished capillary tube, whose length exceeds that of the colon.
3. An artificial pellet is mounted to the capillary glass and the colon is positioned with the pellet in the middle.
4. The capillary glass is then fixed to the bottom of the organ bath by the ends protruding from each colonic opening.
5. Suture silk is used to connect three force transducers (model TST125C; Biopac Systems, Santa Barbara, CA) to the proximal, transverse and distal segments of the colon.
6. Resting tension is initially set at 8 mN and monitored using an MP100 interface and recorded on a PC running Acqknowledge software 3.2.6 (Biopac Systems).
7. After spontaneous colonic migrating motor complexes (CMMCs) are detected, yellow-green light from a 532 nm, 300 mW laser (MGL-III-532, OptoEngine) is shined at 5Hz with a pulsewidth of 30ms for 10-60 seconds in duration, either during  spontaneous CMMCs or in between them.
TITLE: Sequencing of construct
AUTHORS: Pascale Baden, michela.deleidi

[DESCRIPTION]
This protocol describes sequencing of construct.

[STEPS]
SECTION: PCR to amplify region of interest
1.  
      Master mix  1x    H2O  13,3    5x   Colorless Reaction Buffer (Promega, #M3005)  4    10mM   dNTPs (ThermoFisher, #R0182)  0,4    Fw primer   (CMV FW)  0,6    Rv primer   (BGH RV)  0,6    GoTaq   Polymerase (Promega, #M3005)  0,1    Σ  19µl  
 Mix 19 µL MM with 1 µL DNA (50 ng).

SECTION: PCR Program
2.   

SECTION: Sodium acetate precipitation
3. Add 50 µL sodium acetate (1 mL 3 Molarity (M) Na Acetate (Carl Roth) + 24 mL 100% EtOH (VWR Chemicals BDH Prolabo)) to 20 µL PCR product.

SECTION: Sodium acetate precipitation
4. Mix well and centrifuge at 3200 rcf, 45 min, 4 °C.
SECTION: Sodium acetate precipitation
5. Remove supernatant (pat plate on paper).
SECTION: Sodium acetate precipitation
6. Add 100 µL 70% EtOH onto pellet.

SECTION: Sodium acetate precipitation
7. Centrifuge at 3200 rcf, 15 min, 4 °C.

SECTION: Sodium acetate precipitation
8. Remove supernatant (pat plate on paper).
SECTION: Sodium acetate precipitation
9. Add 100 µL 70% EtOH onto pellet.

SECTION: Sodium acetate precipitation
10. Centrifuge at 3200 rcf, 15 min, 4 °C.
SECTION: Sodium acetate precipitation
11. Remove supernatant (pat plate on paper).
SECTION: Sodium acetate precipitation
12. Centrifuge upside down max. 600 rcf, 1 min, 4 °C (top of sample down on tissue paper) to remove EtOH.

SECTION: Sodium acetate precipitation
13. Add 15 µL MilliQ-H2O to pellet and vortex 15-20 min (speed 0-1).

SECTION: Sequencing-PCR
14.  
         1xsample    H2O  5,3µl    5x Terminator Sequencing buffer  3,3µl    BigDye v3.1 (ThermoFisher, #4337455)  1,4µl    Σ  10µl  
 Add 5 µL MM.
SECTION: Sequencing-PCR
15. Add 4 µL DNA from step 13.

SECTION: Sequencing-PCR
16. Add 1 µL primer FW or RV.

SECTION: Sequencing program
17.   

SECTION: Sodium acetate precipitation
18. Add 25 µL sodium acetate to 10 µL PCR product from.

SECTION: Sodium acetate precipitation
19. Mix well and centrifuge at 3200 rcf, 45 min, 4 °C.

SECTION: Sodium acetate precipitation
20. Remove supernatant (pat plate on paper).
SECTION: Sodium acetate precipitation
21. Add 100 µL 70% EtOH onto pellet.

SECTION: Sodium acetate precipitation
22. Centrifuge at  3200 rcf, 15 min, 4 °C.
SECTION: Sodium acetate precipitation
23. Remove supernatant (pat plate on paper).
SECTION: Sodium acetate precipitation
24. Add 100 µL 70% EtOH onto pellet.

SECTION: Sodium acetate precipitation
25. Centrifuge at  3200 rcf, 15 min, 4 °C.
SECTION: Sodium acetate precipitation
26. Remove supernatant (pat plate on paper).
SECTION: Sodium acetate precipitation
27. Centrifuge upside down max. 600 rcf, 1 min, 4 °C (top of sample down on tissue paper) to remove EtOH.

SECTION: Sodium acetate precipitation
28. Add 15 µL MilliQ-H2O to pellet and vortex 15-20min (speed 0-1). 
 

SECTION: Load Sequencing-plate
29. Add 10 µL Hi-Di Formamide (Applied Biosystems).

SECTION: Load Sequencing-plate
30. 7 µL DNA after purification (Step 28).

SECTION: Load Sequencing-plate
31. Store in 4 °C until sequencing.
TITLE: DNA extraction from whole blood and buffy coat using the QIAamp DNA mini kit
AUTHORS: Annika Fendler

[DESCRIPTION]
This protocol is for extraction of total RNA from whole blood, or buffy coat or PBMCs using the QIAamp DNA mini kit.


[BEFORE_START]
Prepare:
AW1: Add 25ml EtOH to new bottle
AW2: Add 30 ml EtOH to new bottle
RNAse I stocks: 100 mg/ml



[GUIDELINES]
This kit is for 400 µL Whole blood or 200 µL Buffy coat


[STEPS]
SECTION: DNA Isolation
2. Transfer 200 µL buffy coat or 400 µL whole blood in 1.5 ml collection tubes

SECTION: DNA Isolation
3. Add 20 µL 40 µL Proteinase K and mix by pipetting up or down

SECTION: DNA Isolation
4. Add 4 µL 8 µL    vortex for 15 s 

SECTION: DNA Isolation
5. Add 200 µL 400 µL  buffer AL, vortex for 15 s 

SECTION: DNA Isolation
8. transfer to DNA Mini Spin Column
SECTION: DNA Isolation
9. max x g, 1 min 
Discard collection tube
SECTION: DNA Isolation
10. Add 500 µL  AW1

SECTION: DNA Isolation
11. max rpm, 1 min 
Discard flow-through

SECTION: DNA Isolation
6. Incubate 10 min @ 56 °C 

SECTION: DNA Isolation
12. Add 500 µL  AW2

SECTION: DNA Isolation
13. max rpm, 3 min 
Discard collection tube
SECTION: DNA Isolation
14. Place column in new 2 ml collection tube
SECTION: DNA Isolation
15. max rpm, 1 min 
Discard collection tube

SECTION: DNA Isolation
16. Place column in new 1.5 ml collection tube
SECTION: DNA Isolation
17. 150 µL 200 µL   

SECTION: DNA Isolation
18. Incubate 5 min @ Room temperature 

SECTION: DNA Isolation
19. max rpm, 1 min 

SECTION: DNA Isolation
20. QC: Qubit    and Tape Station   

SECTION: Preparation of whole blood or buffy coat
1. Select whole blood or buffy coat from biobank list and retrieve from storage location. Ensure that sample is correct by scanning the tube barcode and matching it with the biobank list.
SECTION: Preparation of whole blood or buffy coat
1.1. 
 

SECTION: DNA Isolation
7. Add 200 µL  400 µL  ethanol (96–100%) to the sample, and mix again by pulse-vortexing
for 15 s .
TITLE: Evaluating endocytic rate in cells.
AUTHORS: Marine Houdou, Peter Vangheluwe, Nathalie Jacobs

[DESCRIPTION]
Assess endocytic rate in cells using tagged transferrin (Alexa647) and flow cytometry readout.

[BEFORE_START]
Prepare cell culture medium without FBS and keep it at 37°C.

Prepare Flow Cytometry (FC) buffer made of 1% Albumin Fraction V in DPBS (-/-).

Prepare Eppendorf tubes and FC tubes by labeling them and keeping them on ice. 


[GUIDELINES]
Make sure to treat the cells in medium without FBS. 

[STEPS]
1. Seed cells in 12 wellplate that they reach 70-80% confluency the day of the experiment.
 

2. The day of the experiment, remove cell culture medium and pre-treat the cells in medium without FBS in a final volume of500 µL, for 30 min. The different conditions to consider are: 


2.3. After pre-treatment, keep the plate on ice (4°C) for 15 min .
5. Incubate 20 min.
6. Harvest cells and prepare samples for Flow Cytometry (FC).
2.1. No pre-treatment for:
blank samples 
Alexa647-transferrin-only samples. 

In this case, change cell culture medium to cell culture medium without FBS.
2.2. Pre-treatment with endocytosis inhibitors cocktail made of:
100 µM,
50 µM and 
50 µM.
4. Incubate 20 min 
6.1. Discard medium.
6.2. Wash with 500 µL  Versene or DPBS (-/-).
6.3. Discard Versene or DPBS (-/-). 
6.4. Add 200 µL  0.25 % Trypsin or TrypLE. Incubate 5 min.
6.5.  Add 500 µL  1 % BSA-DPBS (-/-), collect the cells and transfer in Eppendorf tubes.
6.6. Centrifuge5 min  at 2500 rpm and 4°C.
6.7. Discard supernatants and resuspend cell pellet in 250-500 µL µL 1 % Albumin Fraction V in DPBS (-/-), depending on the size of the cell pellet. 
 

6.8. Filter cell suspension through Nylon filter into FC tubes. 
7. Keep on ice until acquisition at the flow cytometer. Record 10,000 live events per sample. 
3. Add Alexa647-Transferrin at 50 µg in each well, except for the blank samples.
TITLE: Removal of FRT flanked antibiotic resistance gene
AUTHORS: Elizabeth Fozo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol: Removal of antibiotic resistance marker with FRT sites using pCP20 plasmids</div></div>

[STEPS]
?. [Protocol based on Datsenko 2000]
Transform with pCP20 following transformation protocol. Rescue at    and spread cells on Amp plates.
30 °C
?. [Protocol based on Datsenko 2000]
Incubate overnight at   .(While cells are growing at   the flippase should be in action and should flip out the resistance marker at FRT sites.
30 °C
30 °C
?. [Protocol based on Datsenko 2000]
Optional additional step: Streak colonies on Amp, incubate at   overnight. Will allow for a longer period of flippase action. (This helps if perform protocol first time without it and get low efficiency of flipping out of antibiotic marker)
30 °C
?. [Protocol based on Datsenko 2000]
Patch multiple colonies (~25) onto LB only, LB + Ab with frt sites (eg.Cm or Kan), and LB+Amp and incubate at   overnight. (pCP20 is a temperature-sensitive plasmid and this will help lose the pCP plasmid.)
42 °C
?. [Protocol based on Datsenko 2000]
Take a colony and serially patch onto the plates, starting with LB only, in the order mentioned above. Incubate overnight at the appropriate temperature.
?. [Protocol based on Datsenko 2000]
Original protocol says to patch on LB last to ensure adequate cells are transferred onto antibiotic plates, but BB suggests LB first.
?. [Protocol based on Datsenko 2000]
Interpretation of Results
?. [Protocol based on Datsenko 2000]
No growth on any antibiotic plate – successful removal of marker and nopCPin cells
?. [Protocol based on Datsenko 2000]
Growth on Amp plates – pCP20 not lost from cellsPerform second isolate streak and incubation at   overnight and repeat steps 3 to 4.
42 °C
?. [Protocol based on Datsenko 2000]
Growth on Ab (to be flipped out) plates – failed removal (see optional step 2a for possible troubleshooting)
TITLE: Oxford Nanopore sequencing and library construction
AUTHORS: Rui Zhang

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a protocol for Oxford Nanopore sequencing and library construction, which was used in the humpback puffer genome sequence.</div></div>

[STEPS]
?. ONT Library preparation and Quality Control
?. After obtaining the qualified DNA, the large size fraction was selected by automated gel electrophoresis (BluePippin).
?. Next, the DNA was treated with the end-repair/dA tailing module.
?. After purification, adapter ligation was performed using ligation sequencing kit (LSK109, Oxford Nanopore Technologies).
?. Finally, DNA library was quantified by Qubit.
?. DNA Sequencing
?. A certain concentration and volume of DNA library was loaded onto a flow cell- PromethION cell R9.4.1, which was then transferred to Nanopore PromethION sequencer for real-time single molecule sequencing.
TITLE: Coating plates 
AUTHORS: Hanqin  Li, Oriol Busquets, Steven Poser, Dirk Hockemeyer, Frank Soldner

[DESCRIPTION]
This protocol describes the process of coating plates using either VTN, Matrigel or Geltrex for use in culturing of feeder-free human pluripotent stem cells (hPSCs)

Protocol overview
Coating plates
    A. VTN
    B. Matrigel
    C. Geltrex

General Notes:
Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs. 


[STEPS]
SECTION: A. VTN
1. Thaw aliquoted VTN on ice. Each 6-well plate needs 60 µl VTN. 

SECTION: A. VTN
4. Incubate in a 37 °C  incubator for 60 min 

SECTION: A. VTN
3. Add the diluted VTN to each well, shake and tilt the plate so the VTN solution covers the entire well.
SECTION: A. VTN
2. For each well of 6-well plate, dilute 10 µl VTN in 1 ml pre-chilled DPBS.
SECTION: B. Matrigel
5. Thaw sufficient Matrigel  on ice to coat plates (100 µl Matrigel/6-well plate)

SECTION: B. Matrigel
6. Pipet 100 µl Matrigel into 6 ml cold DMEM/F12 
SECTION: B. Matrigel
7. Quickly transfer 1 ml/well of diluted Matrigel to 6-well plate.

SECTION: B. Matrigel
8. Distribute Matrigel evenly across the surface.
SECTION: B. Matrigel
9. Incubate plates at 37 °C for 60 min 

 

SECTION: B. Matrigel
10. Remove excess Matrigel and wash with DPBS. 
SECTION: B. Matrigel
11. Immediately plate cells at the desired density.
SECTION: C. Geltrex
12. Thaw sufficient Geltrex on ice to coat plates (120 µl/6-well plate)



SECTION: C. Geltrex
13. Dilute Geltrex 1:50 with cold DMEM/F12 (120 µl Geltrex into 6 ml DMEM/F12)



SECTION: C. Geltrex
14. Quickly transfer 1 ml/well of diluted Geltrex to 6-well plate.



SECTION: C. Geltrex
15. Distribute Geltrex evenly across the surface.
SECTION: C. Geltrex
16. Incubate plates at 37 °C for 60 min 

SECTION: C. Geltrex
17. Remove excess liquid from the Geltrex coated plate. 
SECTION: C. Geltrex
18. Immediately plate cells at the desired density.