eamag/protocols-with-steps huggingface dataset:
TITLE: Transfecting COS-1 cells using FuGENE® 4K Transfection Reagent and cell lysis (M-PER) in a 6-well plate
AUTHORS: Jailyn Izu

[DESCRIPTION]
General protocol for transfecting COS-1 cells with FuGene.

[STEPS]
SECTION: Lysis
9. Aspirate the media from all wells.
SECTION: Lysis
10. Wash each well 3 times with 2 mL ice cold 1X PBS (filtered).

SECTION: Lysis
11. Add M-PER with 1X protease and phosphatase inhibitor to each well (see specific protocols for volume)

Be sure to keep M-PER and samples on ice.

Note: HALT protease and phosphatase inhibitor must be diluted in lysis buffer immediately before use (cannot be prepared the day prior).
SECTION: Lysis
12. Scrape the cells from each well 1 at a time using a cell scraper. Pipette all of the M-PER containing the cell lysate from each well into a clean 1.5 mL micro-centrifuge tube and immediately place the tubes on ice.

SECTION: Lysis
13. Centrifuge the tubes for 10 min at 4 °Cat 10000 rcf.

SECTION: Lysis
14. Collect 100 µL aliquots of the supernatant and store them for future use (lysate that will be used within 1-2 weeks after harvesting can be stored at -20 °C, otherwise store lysates in -80 °C, per Cell Signaling recommendations).

SECTION: Transfection
8. Incubate the plate(s) in a 37 °C incubator with 5% CO2 according to the manufacturer's protocol.
SECTION: Transfection
7. After incubation, pipette 100 µL  of the reaction mixture into the corresponding wells. Tilt the plate(s) back and forth to mix.

SECTION: Transfection
6. While incubating, the DNA with FuGene:Opti-MEM,  aspirate the old media from the cells. Add 1 mL of fresh DMEM + 10% FBS + 1% pen-strep to each well.

SECTION: Transfection
5. Add plasmid constructs to each tube containing FuGene:Opti-MEM (ratio according to the manufacturer's protocol  ). Vortex briefly and incubate at room temperature for 15 min.

SECTION: Transfection
4. Once cells have grown to 50-80% confluence, transfect. In a biosafety cabinet, combine FuGene and Opti-Mem (referred to in the Promega protocol as just "medium," volumes according to the manufacturer's protocol  )  in a1.5 mL eppendorf tube. Vortex gently for 1-2 seconds and incubate at room temperature for 5 min.

SECTION: Seed cells
3. Seed COS-1 cells in a 6-well plate (see specific protocols for seeding density and total volume amounts). Incubate the plated cells in 37 °C with 5% CO2 (see specific protocols for duration).
SECTION: Seed cells
1. Cos1 cell line were purchased from (ATCC # CRL-1650) and cultured in DMEM with GlutaMax  DMEM with GlutaMax (Thermo Fisher Scientific, # 10569010)supplemented with 10% FBS Fetal Bovine Serum, certified # 16000044 and 1%  penicillin-streptomycin (Thermo Fisher Scientific, # 15140122).     
SECTION: Seed cells
2. Cells were maintained at 37 °C in 5% CO2 and split 1:5 every 3-5 days based on the confluence (80-100 %) reached through visual monitoring. Cells were not passaged more than 30 times.
TITLE: ShortestSplitlineAlgorithm
AUTHORS: Jamal  Thomas

[DESCRIPTION]
Takes the US Census results and geographic data and creates electoral districts that are iso-populous (same population in each district). This eliminates gerrymandering since it only uses openly available census data, with an algorithm that can be checked by anybody with a common computer.

[BEFORE_START]
Make sure to install the required libraries and Linux distros. Program requires sudo (root) access so check with your system administrator if elevations of permissions is needed. 

[STEPS]
1. Place   ,   , "MakeFile": 
 
into an new folder. 
and "run":
 
into the folder labeled: "State_Data"
2. Unzip the "State_Data" folder. (Warning: Be sure to only unzip the State_Data folder and nothing else as anything else will interfere with program execution)
3. Compile the "geoproc.cpp" and "block_display.c" program
4. Compile the "run" program in the State_Data folder
3.1. Change permission to execute   ,    and  "MakeFile" by using the following command: 
 


3.2. Compile both programs using the following command:
 

4.1. Change permission using the following command:
 

4.2. Run the "run" script using the following command:
 
OR

 

5. Check for newly-created "images" folder for completed results. (Warning: If images are not present, check log file located in the "State_Data" folder for trouble-shooting help.)
TITLE: BAF_Protocol_012_Lipidomics LC-MS(/MS): Vanquish UPLC and Orbitrap ID-X
AUTHORS: Nicholas Sherman

[DESCRIPTION]
This protocol is the basic LC and MS running parameters for lipids or very hydrophobic molecules. Most are singly charged. Lipids can be more challenging as more mass isoforms exist.

[GUIDELINES]
These are our general settings as a starting point for untargeted lipidomics experiments. Specific adjustments need to be made to meet specific samples or type of data required.

[STEPS]
SECTION: Prepare samples for injection
1. Add 10 uL of Splash Mix standard to the extracted lipids. Dry samples under liquid nitrogen at 40C. 

SECTION: Prepare samples for injection
2. Suspend dried extracted samples with 100 µL  of 1:1 IPA/ACN

SECTION: Prepare samples for injection
3. Mix well using a “figure-eight” movement of the vials in the bench. Transfer the sample to a new vial with borosilicate inserts. 
SECTION: Prepare samples for injection
4. Make sure there is no air in the bottom of the inserts  add the vials into the autosampler of the UPLC Vanquish system.
SECTION: Prepare samples for injection
5. In the sequence setup view of the Xcalibur software include each sample in a row filling with: file name, sample ID, folder directory to save results, directory for the acquisition method, vial position and injection volume. Normally use 10% of each sample for injection.
SECTION: LC parameters 15 min gradient
6. 
Speed limits: draw speed 1 uL/s; dispense speed 1 uL/s.
Injection wash parameters: Wash mode: both; Wash time: 10.0s; Wash speed: 10.0 uL/s.
Column temperature: 55 °C 
Run time: 33 min    
Solvents: %A1: 60% ACN in 10  mM Ammonium Formate with 0.1%FA. 
                %A2: 90% IPA, 10% ACN, 10 mM Ammonium Formate with 0.1% FA.
Upper limit pressure: 1500 bar
Flow = 0.260 ml/min 
Equilibration: -3min, 32% B, curve = 5.
Gradient (all steps curve = 5): 0 -1.5 min 32%B; 4 min 45% B; 5 min 52% B; 8 min 58% B;  11min 66% B; 14min 70%B; 18 min 75%B; 21min 97%B; 25 min 97%B; 25.01 min 32% B, 25.01-33 min 32%B. 
SECTION: General Instrument (Orbitrap ID-X) – Positive and Negative modes
7. Ion Source Type: H-ESI
Spray Voltage Positive 3500V 
Spray Voltage Negative 2500V
Ion Transfer Tube Temperature Temperature 275 °C , Sheath gas 35, Aux Gas 7, Sweep gas 1, Vaporizer Temp Temperature 350 °C
Default charge state: 1 
SECTION: Full scan parameters
8. Scan 200-2000 m/z, resolution 120K, Quadrupole isolation: True, normalized AGC 50%, RF lens 40%, Max. Inj. Time 50ms, 1 microscan, Data Type Profile, Polarity Positive or Negative (create two methods one for Positive acquisition and another for Negative acquisition)
SECTION: MS/MS parameters
9. Min intensity 5E4, Dynamic exclusion - 1 time, 5s, 10ppm, isolation 1.5 m/z, resolution 15K, HCD NCE stepped 25, 30, 35%, Max. Inj. Time 50ms, normalized AGC 50%, 1 microscan, Data Type Centroid.
TITLE: SPRI purification beads
AUTHORS: Tomasz Suchan

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol for preparing SPRI beads 100x cheaper than the commercial AMPure XP.</div><div class = "text-block">Based on DeAngelis et al. (1995) and online protocols:</div><div class = "text-block"><a href="https://s3-us-west-2.amazonaws.com/oww-files-public/1/17/Serapure_bead_recipe.pdf" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://s3-us-west-2.amazonaws.com/oww-files-public/1/17/Serapure_bead_recipe.pdf</span></a></div><div class = "text-block"><a href="https://s3-us-west-2.amazonaws.com/oww-files-public/f/f8/SPRI_buffers_v2_2.pdf" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://s3-us-west-2.amazonaws.com/oww-files-public/f/f8/SPRI_buffers_v2_2.pdf</span></a><span>
 </span></div></div>

[STEPS]
?. [Preparation of the solutions]
Prepare 2.5 M solution of NaCl:place a 50 ml Falcon tube on the scale and tareadd 14.61 g of NaCl to the tubeadd water to 40 mldissolve NaClfill up the tube with water to 50 ml and mix
?. [Preparation of the solutions]
Prepare 50% solution of PEG-8000:place a 50 ml Falcon tube on the scale and tareadd 12.5 g of PEG-8000 to the tubeadd 14 ml of nuclease-free waterclose the tube, and dissolve PEG by mixing vigorously, preferably on an orbital shaker with heatingfill up the tube with water to 25 ml and mix gently by flipping the tube to avoid bubbles
?. [Preparation of the solutions]
Prepare TE buffer by mixing:49.4 ml of molecular-grade water500 ul of 1M Tris solution100 ul of 0.5M EDTA solution
?. [Preparation of the solutions]
Prepare 10% Tween-20:add 900 ul of water to 1.5 ml tubeslowly aspirate 100 ul of Tween-20 with just a tip of the pipette submerged to avoid liquid retention outside of the tip (it is very viscous so take time to aspirate the whole volume)add Tween-20 to the water, mix by pippeting
TITLE: Nanopore (without barcode)
AUTHORS: Hsin-Mao   Wu

[DESCRIPTION]
Nanopore

[BEFORE_START]
1. 上機前先用QBIT測量DNA含量
2. 多樣本用Nanopore (with barcode) protocol
3. 依樣本數數據量決定是否加barcode

[GUIDELINES]
1. 含DNA禁止pipetting, vortex
2. 含DNA用輕彈、輕輕spin-down
3.使用filter tip
4. 多個樣本請放在水域槽或保冷孔盤

[STEPS]
SECTION: Step 1 End-prep.
30. 50 µL 中含有100 fmol DNA之DDW

SECTION: Step 1 End-prep.
0.1. 上NEBiocalculator計算DNA用量
並取出計算好用量的DNA
 

SECTION: Step 1 End-prep.
0.2. ds:mass⇆moles
SECTION: Step 1 End-prep.
0.3. moles→mass
SECTION: Step 1 End-prep.
0.4. 輸入DNA片段長度及DNA fmol
輸出DNA mass(ng)
mass/conc.=DNA用量(μl)
SECTION: Step 2 Adapter Ligation
5. 取Step 1之60 µL  產物
SECTION: Step 1 End-prep.
2. 依序加入7 µL  End Repair & A-Tailing Buffer, 3 µL  End Repair & A-Tailing Enzyme Mix
SECTION: Step 1 End-prep.
1. 加入DDW至50 µL 

SECTION: Step 1 End-prep.
3. 加完後vortex、離心數秒
SECTION: Step 1 End-prep.
4. 放進水浴槽：
 

[nanopore_step1]
sample要均分2管(每管30μl) 放在機器裡正中間的槽
[edit]確認流程
[run]之前改sample量
SECTION: Step 2 Adapter Ligation
6. 依序加入5 µL AMX, 5 µL DI Water, 30 µL Ligation Buffer, 10 µL DNA Ligase

Ligation Buffer 輕彈至完全混合
DNA Ligase先不要拿
SECTION: Step 2 Adapter Ligation
7. 放進水浴槽：
 

[Nanopore_step2]
sample均分2管(每管55μl)
[edit]確認流程
[run]之前改sample量
SECTION: Step 3 DNA library clean-up
8. 取Step 2之110 µL 產物

SECTION: Step 3 DNA library clean-up
9. 加入44 µL AMPure XP Beads
 


SECTION: Step 3 DNA library clean-up
10. 靜置5 min 

SECTION: Step 3 DNA library clean-up
11. 離心數秒
SECTION: Step 3 DNA library clean-up
12. 放置於磁座上，等待磁鐵吸附Beads
SECTION: Step 3 DNA library clean-up
13. 移除上清液
 

SECTION: Step 3 DNA library clean-up
14. 加入250 µL LFB

SECTION: Step 3 DNA library clean-up
15. 持續輕彈離心管3 min 
 


SECTION: Step 3 DNA library clean-up
16. 放在磁座上 等待磁鐵吸附beads
SECTION: Step 3 DNA library clean-up
17. 移除上清液，7~9步驟再重複一次
 

SECTION: Step 3 DNA library clean-up
18. 加入15 µL Elution Buffer

SECTION: Step 3 DNA library clean-up
19. 輕彈將磁珠彈離管壁
SECTION: Step 3 DNA library clean-up
20. 離心數秒；放置磁座上
 
等待磁鐵吸附beads
SECTION: Step 3 DNA library clean-up
21. 靜置10 min 至Room temperature 

SECTION: Step 3 DNA library clean-up
22. 取1 µL 測QBIT

SECTION: Step 4 Priming and loading
23.
TITLE: 18 Monitoring in living bacterial cells by UV-Vis spectroscopy
AUTHORS: TJUSLS China

[STEPS]
?. Pipet 5μL NDM-28a BL21(DE3) glycerol bacteria into 5ml LB medium, and 2.5μL kanamycin is added. Incubate aiming bacterial liquid at 37°C until its OD600 reach 0.5-0.6 then add inducer IPTG.
?. Centrifuge bacterial liquid and add phosphate buffer to resuspend bacterial precipitation, then centrifuge again and discard phosphate buffer. Repeat 3 times to wash precipitate.
?. Mix bacterial precipitate in phosphate buffer in incubation, and dilute it. OD600 of the bacterial liquid used for next measurement is 0.15.
?. UV-Vis test I. Test one experimental group together with 3 different controls. Record the absorption value every 300 seconds, 12 times in total.95μL bacterial liquid which express target protein, 5μL cefazolin(final concentration is 250μM);95μL beta-lactamase(final concentration is decided by characteristic of enzyme), 5μL cefazolin(final concentration is 250μM);95μL bacterial liquid which is transferred with blank vector, 5μL cefazolin(final concentration is 250μM);95μL phosphate buffer, 5μL cefazolin(final concentration is 250μM). Then plot the UV-vis spectroscopy with time.
?. Establish a system for the determination of viable bacteria.95μL bacterial liquid with different induction time and OD value was mixed with 5μL cefazolin(final concentration is 250μM)to determine the optimal induction time and OD. Record the absorption value every 300 seconds, 24 times in total.
?. UV-Vis test II. Test the UV absorption peak in 273nm(cefazolin), 307nm(meropenem), 300nm(faropenem), 360nm(tetracycline)95μL bacterial liquid which express target protein, 5μL cefazolin(final concentration is 250μM);95μL bacterial liquid which express target protein, 5μL meropenem(final concentration is 250μM);95μL bacterial liquid which express target protein, 5μL faropenem(final concentration is 250μM);95μL bacterial liquid which express target protein, 5μL tetracycline(final concentration is 250μM).
?. UV-Vis test III.94μL bacterial liquid which express target protein, 5μL cefazolin(final concentration is 250μM), 1μL inhibitor;94μL bacterial liquid which express target protein, 5μL cefazolin(final concentration is 250μM), 1μL inhibitor’s solvent (100% DMSO);94μL phosphate buffer, 5μL cefazolin(final concentration is 250μM), 1μL inhibitor's solvent (100% DMSO);94μL phosphate buffer, 5μL cefazolin’s solvent, 1μL inhibitor's solvent (100% DMSO).Test a series of inhibitor’s concentration as a gradient and test 5 parallel control. Then calculate the inhibition rate for each concentration as equation 1, and plot IC50 curve.Equation 1: Inhibition rate% = 100*（1-（[St]-[Si]）/([St]-[So])）[St] = Initial absorption value of antibiotics[Si] = Terminated absorption value of antibiotics with the addition of inhibitors[So] = Terminated absorption value of antibiotics without the addition of inhibitors
TITLE: Methodologies of chosen playlist selection for people living with dementia: A Systematic Review Protocol
AUTHORS: bethanyroseevison, Nicolas Farina

[DESCRIPTION]
Abstract

Background: To further personalise and refine playlist collection methodologies for people with dementia, current utilised methodologies must be reviewed and critically analysed. However, there is limited transparency of some of these approaches. To remedy this current gap in literature more focussed research into the methodologies used in creating these music playlists is needed (Garrido et al., 2017). This protocol is for a systematic review that aims to identify the methodologies currently utilised in the development of music playlists for people with dementia and will also explore and analyse the effectiveness of these methods on dementia related outcomes. 

Methods: This protocol has been written in line with the Preferred Reporting Items for Systematic Reviews and Meta-analyses Protocol (PRISMA) guidelines. Systematic searches will be used to identify the efficacy of methodologies used to collect chosen playlists for people living with dementia. These searches will be conducted across PubMed, PsychInfo, SCOPUS, and Music Periodicals database. Titles, abstracts and keywords will be screened with Rayyan.ai software and risk of bias will be assessed using the mixed methods appraisal tool (MMAT). 

Results: Studies will be reported as descriptive data and summarised narratively. Data and information on specific methodologies will be grouped together. Depending on the variability of methods, this might occur through grouping methodologies into broader themes (e.g., informant-based questionnaire playlists). Identifying and critically analysing the efficacy and acceptability of playlist collection methodologies will further understanding and future refine of further tailored and user-friendly playlist collection methods for people with dementia.

Discussion: Findings from the systematic review will help aid the refine and development of new methodologies for playlist collection in people with dementia, so that it is not only effective in improving outcomes and objectives but also acceptable and feasible. 

Keywords: Dementia, music, chosen playlists, individual music listening.

[STEPS]
1.
TITLE: Neural progenitor banking
AUTHORS: Celeste Karch, Rita Martinez, Jacob Marsh

[STEPS]
?. Upon reaching at least 85% confluency, harvest neural progenitor cells as described in protocol below. Neural progenitor expansion protocol
?. Add equal volumes of NIM and 2x neural freezing medium to the NPC cell suspension for a final 1 x 106 cells/mL.
?. Gently mix solution and distribute  into sterile cryovials. Store cryovials in Styrofoam containers atfor  and then transfer to liquid nitrogen for long-term storage.
1 ml
?. Perform a cell count  in of NIM using a hemacytometer.
3 ml
TITLE: Dementia post-diagnostic support in UK rural communities: experiences of people living with dementia, informal caregivers, and healthcare professionals. A systematic review protocol.
AUTHORS: Dannielle Bilkey, Nicolas Farina, Ben Hicks

[DESCRIPTION]


People living with dementia in rural areas experience numerous barriers to accessing post-diagnostic support. The aim of this systematic review is to identify and describe the experiences, barriers and provision of post-diagnostic support in UK rural areas; from the perspective of people living with dementia, healthcare professionals and informal caregivers. Systematic searches will be conducted in the following databases; SCOPUS, PubMed, PsychINFO and CINAHL Plus. Systematic review tool Rayyan.ai will be used to screen titles and abstracts, prior to full-text review. Following data extraction, The Critical Appraisal Skills Programme (CASP) will be used to appraise the quality of studies and assess risk of bias. The data will be deductively analysed through the lens of the Candidacy Framework's 6 dimensions, with a secondary inductive analysis capturing any themes that fall outside of the framework. Understanding the rural barriers and experiences of post-diagnostic support will allow researchers and stakeholders to develop and optimise specially tailored dementia interventions in line with the needs of people residing in UK rural communities. 




[STEPS]
TITLE: Tissue processing and freezing after surgery
AUTHORS: Bettina Ergün

[DESCRIPTION]
The aim of this protocol is to document the processing of fresh tissue after surgery.
 
The collected tissue must also be documented in the files which are linked down below
 
The protocol descibes in detail how to process the resulting tissue from following protocols:

Gewebesammlung Frischgewebe Zystektomie
Gewebesammlung Frischgewebe Prostatektomie
Gewebesammlung Frischgewebe Nephrektomie

in the current version.







[STEPS]
SECTION: Sample processing in the lab
1. 
Fresh tissue should be processed on the same day if possible, but can be stored at 4°C for a maximum of overnight.
To reduce pathogens, the tissue should be incubated for 60 min atRoom temperature 

SECTION: Sample processing in the lab
2. 
 
 

 

SECTION: Sample processing in the lab
3. 
 

SECTION: Sample processing in the lab
4. 

The tissue is divided in the lab into pieces with an edge length of 1-2 mm.
Mix the fragments of a tumor piece so that each aliquot is as representative as possible of the tumor area.
SECTION: Sample processing in the lab
5. 16 (decision from June 29, 2023; previously: 8) fragments are stored in 1 ml of freezing medium in a cryotube.
If there are enough fragments for more than 4 cryotubes, more fragments can be combined in one tube. A maximum of 5 cryotubes of each entity are frozen.

Cryo tubes should be labelled as follows and documented in the N2 binder:
Tissue ID,T1/T2 resp. N1/N2...
No. of Pieces
Date
SECTION: Sample processing in the lab
6. Freeze samples in Mr. Frosty overnight.
SECTION: Documentation
7. Store patient data and enter the corresponding liquid number:
https://charitede.sharepoint.com/:x:/r/sites/UroForsch/_layouts/15/doc2.aspx?sourcedoc=%7B1282209D-5A7C-4998-A577-145386116E54%7D&file=Biobank_Gewebe_ab01012019.xlsx&action=default&mobileredirect=true
SECTION: Documentation
8. Enter the corresponding tissue number in the liquid collection list:
https://charitede.sharepoint.com/:x:/r/sites/UroForsch/_layouts/15/Doc.aspx?sourcedoc=%7BCE8CD395-BFB0-4B2C-8220-37FB802726A7%7D&file=Biobanking-lfdListe_ab190311.xlsx&action=default&mobileredirect=true
SECTION: Documentation
9. Please add pictures of:
the orgin of the sample in the organ (if avaible)
each tissue piece that has been taken before cutting the fragments. The picture should include the tissue ID (in the picture)
the documentation sheet
TITLE: DNA extraction protocol (Salting out) Modified
AUTHORS: Afaq M.M. Niyas, Caterina Villari

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes how to extract DNA with modified salting out method.</div><div class = "text-block"><span>This protocol is part of the Bark Beetle Mycobiome (BBM) Research Coordination Network. For more information on the BBM international network: Hulcr J, Barnes I, De Beer ZW, Duong TA, Gazis R, Johnson AJ, Jusino MA, Kasson MT, Li Y, Lynch S, Mayers C, Musvuugwa T, Roets F, Seltmann KC, Six D, Vanderpool D, & Villari C. 2020. Bark beetle mycobiome: collaboratively defined research priorities on a widespread insect-fungus symbiosis. Symbiosis 81: 101–113 </span><a href="https://doi.org/10.1007/s13199-020-00686-9" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://doi.org/10.1007/s13199-020-00686-9</span></a><span>.</span></div></div>

[STEPS]
TITLE: Use of Health Improvement Card by Chinese physical therapy students: A pilot study
AUTHORS: Alice Jones, XB Wu, YW Bai, Jia Han, Elizabeth Dean

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the methods for a study aimed to investigate perceptions of Chinese physical therapy students on the use of the Health Improvement Care (HIC) as a clinical tool to assess lifestyle behaviors/attributes and provide lifestyle education advice to others. Title of the study: Use of Health Improvement Card by Chinese physical therapy students: A pilot study. Xubo Wu, Alice YM Jones, Yiwen Bai, Jia Han, Elizabeth Dean.</div></div>

[STEPS]
?. Physical therapy students were recruited from two universities in Shanghai, China.
?. Introduction of the Health Improvement Card (HIC) to physical therapy students from two universities in Shanghai via a standardized 45-min tutorial. Content of the tutorial follows the User Guide for administering the HIC; both of which were developed by the World Health Professions Alliance (www.whpa.org/ncd_health_improvement_card_professionals.pdf).
?. Students were asked to complete the 2-page Chinese version of the HIC (Appendix 1). Height and weight of the students were self-reported; waist circumference and blood pressure were measured.
?. Students completed a questionnaire on their perceptions of the use and usefulness of the HIC. The questionnaire consisted of 7 statements and asked students to rate their level of agreement with each statement using a 4-point Likert scale. (Appendix 2).
?. Students invited a friend or relative to complete the HIC with the student serving in the role of the health professional. Completed HICs were returned to the investigators within one week.
?. Students completed a second questionnaire with three open-ended questions that were designed to obtain feedback about the positive aspects of administering the HIC and any challenges encountered in their role as a health professional, when administering the Card to their friends/relatives. (Appendix 3).