HIV Cure Protocol: Bench Quick Reference

Equipment Checklist

Day 1 Essentials

Equipment	Est. Cost	Vendor Examples
BSL-2 biosafety cabinet	Facility	Thermo, Baker
CO2 incubator (37C, 5%)	$5-10K	Thermo, Eppendorf
Flow cytometer (4+ color)	$50-150K	BD, Beckman, Cytek
Real-time qPCR	$25-40K	Bio-Rad, Applied Biosystems
Electroporator	$15-25K	Neon (Thermo), Lonza
Plate reader (lum + abs)	$15-30K	BioTek, Tecan
-80C freezer	$8-15K	Thermo, Stirling
LN2 storage	$5-10K	Thermo, MVE
Centrifuges	$5-15K	Eppendorf, Beckman

Phase 2 Additions

Equipment	Est. Cost
ddPCR system	$80-120K
Cell sorter (FACS)	$150-300K
Automated liquid handler	$50-150K

Reagent Shopping List

Cell Culture

Reagent	Catalog Example	Notes
RPMI 1640	Gibco 11875	T cell base
FBS	Gibco 10437	Heat-inactivate, lot test
Pen/Strep	Gibco 15140	100 U/mL working
GlutaMAX	Gibco 35050	2 mM working
HEPES	Gibco 15630	10-25 mM optional
IL-2	Peprotech 200-02	50 U/mL for T cells
IL-7	Peprotech 200-07	5 ng/mL for resting
M-CSF	Peprotech 300-25	50 ng/mL for macrophages
anti-CD3 beads	Miltenyi 130-091-441	T cell activation
anti-CD28 beads	Miltenyi 130-091-441	T cell activation

HIV-Specific (NIH AIDS Reagent Program)

Reagent	Catalog	Purpose
J-Lat 10.6	ARP-9849	Latency model
ACH-2	ARP-349	Chronic infection
MT-2	ARP-237	Permissive cells
HIV-1 NL4-3	ARP-114	Clade B reference
HIV-1 BaL	ARP-510	R5-tropic
p24 antibody (KC57)	Beckman 6604667	Flow cytometry
VRC01 bNAb	ARP-12033	Env detection

Molecular Biology

Reagent	Purpose
dCas9-KRAB plasmid	Addgene (multiple options)
Synthetic sgRNAs	IDT or Synthego
Lipofectamine 3000	Thermo L3000015
RNA extraction kit	Qiagen 74104
cDNA synthesis	Invitrogen 18080051
SYBR Green master mix	Bio-Rad 1725271
ddPCR supermix	Bio-Rad 1863024
ChIP kit	Active Motif 53040
H3K9me3 antibody	Abcam ab8898

Flow Cytometry Antibodies

Target	Clone	Fluorophore	Purpose
CD4	RPA-T4	APC	T cell ID
CD3	UCHT1	FITC	T cell ID
CD8	SK1	PE	Exclusion
CD45RA	HI100	BV421	Memory subsets
CCR7	G043H7	PE-Cy7	Memory subsets
CD14	M5E2	APC	Monocyte/Mac
CD68	Y1/82A	PE	Macrophage
Annexin V	-	FITC	Apoptosis
Zombie Aqua	-	-	Viability

Standard Operating Procedures

SOP-001: J-Lat Reactivation Assay

Timing: 2 days

Materials:

J-Lat 10.6 (passage <20)
Complete RPMI (RPMI + 10% FBS + P/S + GlutaMAX)
TNF-a stock: 10 ug/mL -> working 10 ng/mL
PMA stock: 10 mM -> working 10 nM
Zombie Aqua viability dye

Step	Action	Time
1	Seed 2x10^5 cells/well (24-well, 500 uL)	0h
2	Add TNF-a (10 ng/mL final) or vehicle	0h
3	Incubate 37C, 5% CO2	24h
4	Harvest, wash PBS	24h
5	Stain viability (1:1000, 10 min RT)
6	Wash, resuspend FACS buffer
7	Acquire: Live -> Singlets -> GFP+

QC Criteria

Vehicle: <5% GFP+
TNF-a: >30% GFP+
Viability: >80%

SOP-002: Primary CD4+ T Cell Isolation

Timing: 4-5 hours

Materials:

Fresh leukapheresis or buffy coat
Ficoll-Paque (GE 17-1440)
CD4+ T Cell Isolation Kit (Miltenyi 130-096-533)
MACS LS columns + separator

Step	Action
1	Dilute blood 1:1 with PBS
2	Layer 35 mL over 15 mL Ficoll (50 mL tube)
3	Centrifuge 400g, 30 min, brake OFF
4	Harvest cloudy PBMC interface
5	Wash 2x PBS
6	Count, resuspend 10^8/mL
7	Add isolation cocktail per kit
8	Incubate 5 min, 4C
9	Add magnetic beads
10	Incubate 10 min, 4C
11	Pass through LS column on magnet
12	Collect flow-through (CD4+ enriched)
13	Verify purity by flow

QC Criteria

Purity: >95% CD4+ CD3+
Viability: >95%
Recovery: >70%

SOP-003: HIV Infection of Primary Cells

Timing: Day 0 infection, Day 3+ for latency

Materials:

Activated CD4+ T cells (day 3 post-activation)
HIV-1 NL4-3 virus stock (titered, store -80C)
Polybrene stock: 4 mg/mL -> working 4 ug/mL

Step	Action
1	Calculate virus volume for MOI 0.1
2	Resuspend 10^6 cells in 200 uL
3	Add virus + polybrene
4	Spinoculate: 1200g, 2h, 25C
5	Wash 3x complete media
6	Resuspend 10^6/mL + IL-2
7	Culture 3d, check infection by p24 flow
8	For latency: remove beads, switch to IL-7
9	Rest 7-14d

QC Criteria

Day 3: 10-30% p24+ by flow
Post-resting: <5% spontaneous p24+
Reactivatable: >20% p24+ after PHA/PMA

SOP-004: Electroporation (Neon System)

Timing: 1-2 hours

Materials:

Neon Transfection System
Buffer R (Neon kit)
dCas9-KRAB mRNA or plasmid
Synthetic sgRNAs

Optimized Settings

Cell Type	Voltage	Pulse Width	Pulses
J-Lat	1400V	10ms	3
Primary CD4+	1600V	10ms	3
HEK293T	1100V	20ms	2

Step	Action
1	Harvest cells, wash PBS
2	Resuspend 10^6 cells in 100 uL Buffer R
3	Add mRNA (1-2 ug) + sgRNAs (100 pmol each)
4	Load 100 uL Neon tip
5	Electroporate with optimized settings
6	Immediately transfer to pre-warmed media
7	Culture 4-24h before analysis

QC Criteria

Viability post-electroporation: >70%
Confirm dCas9 expression (if tagged)

SOP-005: ChIP-qPCR for H3K9me3

Timing: 3 days

Materials:

SimpleChIP kit (CST 9003) or equivalent
H3K9me3 antibody (Abcam ab8898)
IgG control
LTR primers

LTR Primer Sequences

LTR-F: 5'-TGTGTGCCCGTCTGTTGTGT-3'
LTR-R: 5'-GAGTCCTGCGTCGAGAGAGC-3'
                

Day	Step	Action
1	1	Cross-link: 1% formaldehyde, 10 min, RT
	2	Quench: 125 mM glycine, 5 min
	3	Lyse per kit protocol
	4	Sonicate to 200-500 bp
	5	Clear lysate (spin 12,000g, 10 min)
	6	Save 10% as input
	7	Add antibody + beads, rotate O/N 4C
2	8	Wash per kit protocol
	9	Elute and reverse cross-links
3	10	Purify DNA
	11	qPCR with LTR primers
	12	Calculate % input or fold over IgG

QC Criteria

Sonication: check 200-500 bp by gel
Positive control locus enriched
IgG background <0.1% input

SOP-006: p24 ELISA

Timing: 5-6 hours

Materials:

p24 ELISA kit (PerkinElmer NEK050)
Cell supernatant samples
Standards (provided)

Step	Action	Time
1	Add standards and samples to plate	-
2	Incubate	2h RT
3	Wash 4x	-
4	Add detection antibody	1h RT
5	Wash 4x	-
6	Add substrate	15-30 min
7	Stop reaction	-
8	Read 450 nm	-
9	Calculate from standard curve	-

QC Criteria

Standard curve R2 >0.99
CV duplicates <15%
Samples in linear range

Troubleshooting Quick Reference

Problem

Check

Fix

Low transfection

Cell viability pre-transfection

Use healthier cells

Variable reactivation

TNF-a activity

Aliquot, avoid freeze-thaw

High flow background

Compensation

Run single-stain controls

ChIP signal low

Sonication

Verify 200-500 bp fragments

p24 ELISA variable

Sample handling

Clarify supernatant

Primary cells dying

Media age, cytokines

Fresh media, add IL-7/IL-2

sgRNA not working

Sequence, delivery

Verify sequence, increase dose

dCas9 toxicity

Expression level

Reduce mRNA dose, shorten time

Critical Reagent Storage

Reagent	Storage	Stability
HIV stocks	-80C	Years
TNF-a aliquots	-20C	6 months
PMA aliquots	-20C (DMSO)	1 year
sgRNAs	-20C	1 year
mRNA	-80C	6 months
Antibodies	4C	Per manufacturer
ELISA kits	4C	Check expiry

Vendor Quick Links

NIH AIDS Reagent Program Addgene (dCas9 plasmids) IDT (sgRNAs) Synthego (sgRNAs) Peprotech (cytokines) Miltenyi (cell isolation)